Note: Descriptions are shown in the official language in which they were submitted.
CA 02528202 1999-10-21
DEiNLA,NDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECIESTLETOINIE~DE
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME OF
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02528202 1999-10-21
PROCESS
Backaround of the Invention
Field of the Invention
This invention relates to a process for producing and recovering
heterologous polypeptides from bacterial cells. More particularly, this
invention relates to a process wherein recovery of soluble or aggregated
recombinant heterologous polypeptides from bacterial cytoplasm and periplasm
is facilitated or increased.
Description of Related Disclosures
Escherichia coli has been widely used for the production of heterologous
proteins in the laboratory and industry. E. coli does not generally excrete
proteins to the extracellular medium apart from colicins and hemolysin
(Pugsley
and Schwartz, Microbiology, 1$: 3-38 (1985)). Heterologous proteins expressed
by E. coil may accumulate as soluble product or insoluble aggregates. See
Figure 1 herein. They may be found intracellularly in the cytoplasm or be
secreted into the periplasm if preceded by a signal sequence. How one proceeds
initially in the recovery of the products greatly depends upon how and where
the product accumulates. Generally, to isolate the proteins, the cells may be
subjected to treatments for periplasmic extraction or be disintegrated to
release trapped products that are otherwise inaccessible.
The conventional isolation of heterologous polypeptide from gram-negative
bacteria poses problems owing to the tough, rigid cell walls that surround
these cells. The bacterial cell wall maintains the shape of the cell and
protects the cytoplasm from osmotic pressures that may cause cell lysis; it
performs these functions as a result of a highly cross-linked peptidoglycan
(also known as murein) backbone that gives the wall its characteristic
rigidity. A recent model described the space between the cytoplasmic and outer
membranes as a continuous phase filled with an inner periplasmic
polysaccharide
gel that extends into an outer highly cross-linked peptidoglycan gel (Hobot et
al., J. Bact., 10: 143 (1984)). This peptidoglycan sacculus constitutes a
barrier to the recovery of any heterologous polypeptide not excreted by the
bacterium into the medium.
To release recombinant proteins from the E. coil periplasm, treatments
involving chemicals such as chloroform (Ames et al., J. Bacteriol., 160: 1181-
1183 (1984)), guanidine-HC1, and Triton X-100 (Naglak and Wang, Enzyme Microb.
Technol., 122: 603-611 (1990)) have been used. However, these chemicals are
not
inert and may have detrimental effects on many recombinant protein products or
subsequent purification procedures. Glycine treatment of E. coli cells,
causing permeabilization of the outer membrane, has also been reported to
release the periplasmic contents (Ariga et al., J. Ferro. Bioena., 68: 243-246
(1989)). These small-scale periplasmic release methods have been designed for
specific systems. They do not translate easily and efficiently and are
generally unsuitable as large-scale methods.
-1-
CA 02528202 1999-10-21
The most widely used methods of periplasmic release of recombinant
protein are osmotic shock (Nosal and Heppel, J. Biol. Chem., 241: 3055-3062
(1966); Neu and Heppel, J. Biol. Chem., 240: 3685-3692 (1965)), hen
eggwhite(HEW)-lysozyme/ethylenediamine tetraacetic acid (EDTA) treatment (Neu
and Heppel, J. Biol. Chem., 239: 3893-3900 (1964); Witholt et al., Biochim.
Biovhvs. Acta, 443: 534-544 (1976); Pierce et al., ICheme Research Event, -2-
995-997 (1995)), and combined HEW-lysozyme/osmotic shock treatment (French et
al., Enzyme and Microb. Tech., 19: 332-338 (1996)). Typically, these
procedures include an initial disruption in osmotically-stabilizing medium
followed by selective release in non-stabilizing medium. The composition of
these media (pH, protective agent) and the disruption methods used
(chloroform,
HEW-lysozyme, EDTA, sonication) vary among specific procedures reported. A
variation on the HEN-lysozyme/EDTA treatment using a dipolar ionic detergent
in place of EDTA is discussed by Stabel et al., Veterinary Microbiol., 9U: 307-
314 (1994). For a general review of use of intracellular lytic enzyme systems
to disrupt H. cola, see Dabora and Cooney in Advances in Biochemical
Engineering/Biotechnology, Vol. 43, A. Fiechter, ed. (Springer-Verlag: Berlin,
1990), pp. 11-30.
Conventional methods for the recovery of recombinant protein from the
cytoplasm, as soluble protein or retractile particles, involved disintegration
of the bacterial cell by mechanical breakage. Mechanical disruption typically
involves the generation of local cavitation in a liquid suspension, rapid
agitation with rigid beads, sonication, or grinding of cell suspension
(Bacterial Cell Surface Techniques, Hancock and Poxton (John Wiley & Sons Ltd,
1988), Chapter 3, p. 55). These processes require significant capital
investment and constitute long processing time.
HEW-lysozyme acts biochemically to hydrolyze the peptidoglycan backbone
of the cell wall. The method was first developed by Zinder and Arndt, Proc.
Natl. Acad. Sci. USA, 4a: 586-590 (1956), who treated S. coli with egg albumin
(which contains HEW-lysozyme) to produce rounded cellular spheres later known
as spheroplasts. These structures retained some cell-wall components but had
large surface areas in which the cytoplasmic membrane was exposed.
U.S. Pat. No. 5,169,772 discloses a method for purifying heparinase from
bacteria comprising disrupting the envelope of the bacteria in an osmotically-
stabilized medium, e.g., 20% sucrose solution using, e.g., EDTA, lysozyme, or
an organic compound, releasing the non-heparinase-like proteins from the
periplasmic space of the disrupted bacteria by exposing the bacteria to a low-
ionic-strength buffer, and releasing the heparinase-like proteins by exposing
the low-ionic-strength-washed bacteria to a buffered salt solution.
There are several disadvantages to the use of the HEW-lysozyme addition
for isolating periplasmic proteins. The cells must be treated with EDTA,
detergent, or high pH, all of which aid in weakening the cells. Also, the
method is not suitable for lysis of large amounts of cells because the
lysozyme
-2-
CA 02528202 1999-10-21
addition is inefficient and there is difficulty in dispersing the enzyme
throughout a large pellet of cells.
Many different modifications of these methods have been used on a wide
range of expression systems with varying degrees of success (Joseph-Liazun et
ai., Gene, SAC: 291-295 (1990); Carter et al., Bio/Technoloav, 10: 163-167
(1992)). Although these methods have worked on a laboratory scale, they
involve too many steps for an efficient large-scale recovery process.
Efforts to induce recombinant cell culture to produce lysozyme have been
reported. EP 155,189 discloses a means for inducing a recombinant cell culture
to produce lysozymes, which would ordinarily be expected to kill such host
cells by means of destroying or lysing the cell wall structure. Russian Pat.
Nos. 2043415, 2071503, and 2071501 disclose plasmids and corresponding strains
for producing recombinant proteins and purifying water-insoluble protein
agglomerates involving the lysozyme gene. Specifically, the use of an operon
consisting of the lysozyme gene and a gene that codes for recombinant protein
enables concurrent synthesis of the recombinant protein and a lysozyme that
breaks the polysaccharide membrane of E. coli.
U.S. Pat. No. 4,595,658 discloses a method for facilitating
externalization of proteins transported to the periplasmic space of E. coll.
This method allows selective isolation of proteins that locate in the
periplasm
without the need for lysozyme treatment, mechanical grinning, or osmotic shock
treatment of cells. U.S. Pat. No. 4,637,980 discloses producing a bacterial
product by transforming a temperature-sensitive lysogen with a DNA molecule
that codes, directly or indirectly, for the product, culturing the
transformant
under permissive conditions to express the gene product intracellularly, and
externalizing the product by raising the temperature to induce phage-encoded
functions. JP 61-257931 published November 15, 1986 discloses a method for
recovering IL-2 using HEW-lysozyme. Asami et al., J. Ferment. and Bioeng., 83:
511-516 (1997) discloses synchronized disruption of S. coil cells by T4 phage
infection, and Tanji et al., J. Ferment. and Bioena., 85: 74-78 (1998)
discloses controlled expression of lysis genes encoded in T4 phage for the
gentle disruption of S. coli cells.
The development of an enzymatic release method to recover recombinant
periplasmic proteins suitable for large-scale use ~s reported by French et
al.,
Enzyme and Microbial Technoloav, : 332-338 (1996). This method involves
resuspension of the cells in a fractionation buffer followed by recovery of
the
periplasmic fraction, where osmotic shock immediately follows lysozyme
treatment. The effects of overexpression of the recombinant protein, S.
thermoviolaceus a-amylase, and the growth phase of the host organism on the
recovery are also discussed.
In a 10-kiloliter-scale process for recovery of IGF-I polypeptide (Hart
et al., Bio/Technoloav, U: 1113 (1994)), the authors attempted the typical
isolation procedure involving a mechanical cell breakage step followed by a
-3-
CA 02528202 1999-10-21
centrifugation step to recover the solids. The results were disappointing in
that almost 40% of the total product was lost to the supernatant after three
passes through the Gaulin homogenizer. Hart et al., Bio/Technology 12: 1113
(1994). See Fig. 2 herein. Product recovery was not significantly improved
even when the classical techniques of EDTA and HEW-lysozyme additions were
employed.
While HEW-lysozyme is the only practical commercial lysozyme for large-
scale processes, lysozyme is expressed by bacteriophages upon infection of
host
cells. Lysis of S. coli, a natural host for bacteriophages, for example the
T4 phages, requires the action of two gene products: e and t. Gene e encodes
a lysozyme (called T4-lysozyme for the T4 phage) that has been identified as
a muramidase (Tsugita and Inouye, J. Biol. Chem., 243: 391 (1968)), while gene
t seems to be required for lysis, but does not appear to have lysozyme
activity. Gene t is required for the cessation of cellular metabolism that
occurs during lysis (Mukai et al., Vir., 33: 398 (1967)) and is believed to
degrade or alter the cytoplasmic membrane, thus allowing gene product a to
reach the periplasm and gain access to the cell wall (Josslin, Vir., 40: 719
(1970)). Phage are formed by gene t- mutants, but lysis of the H. coli host
does not occur except by addition of chloroform (Josslin, supra). Wild-type
T4-lysozyme activity is first detected about eight minutes after T4 infection
at 37 C, and it increases through the rest of the infection, even if lysis
inhibition is induced. In the absence of secondary adsorption, cells infected
by gene e mutants shut down progeny production and metabolism at the normal
time, but do not lyse (Molecular Genetics of Bacteriochaae T4, J.D. Karam, ed.
in chief (American Society for Microbiology, Washington DC, ASM Press, 1994),
p. 398).
Recovery of insoluble IGF-X using T4-lysozyme was disclosed on October
28, 1997 at the "Separation Technology VII meeting entitled `Separations for
Clean Production'" in Davos, Switzerland, sponsored by the Engineering
Foundation.
Upon cell lysis, genomic DNA leaks out of the cytoplasm into the medium
and results in significant increase in fluid viscosity that can impede the
sedimentation of solids in a centrifugal field. In the absence of shear forces
such as those exerted during mechanical disruption to break down the DNA
polymers, the slower sedimentation rate of solids through viscous fluid
results
in poor separation of solids and liquid during centrifugation. Other than
mechanical shear force, there exist nucleolytic enzymes that degrade DNA
polymer.
In S. coli, the endogenous gene endA encodes for an endonuclease
(molecular weight of the mature protein is approx. 24.5 kD) that is normally
secreted to the periplasm and cleaves DNA into oligodeoxyribonucleotides in an
endonucleolytic manner. It has been suggested that endA is relatively weakly
expressed by S. coli (Wackernagel et al., Gene, 154: 55-59 (1995)).
-4-
CA 02528202 1999-10-21
For controlling cost of goods and minimizing process time, there is a
continuing need for increasing the total recovery of heterologous polypeptide
from cells. At large scale, there is a significant incentive to avoid
mechanical cell breakage to release the soluble or aggregated recombinant
polypeptide from the cytoplasmic and periplasmic compartments and to condition
the lysate for efficient product recovery in the subsequent step.
Summary of the Invention
Accordingly, this invention provides processes using biochemical
disruption to recover both soluble and insoluble heterologous product from
bacterial cells.
In one aspect the present invention provides a process for recovering a
heterologous polypeptide from bacterial cells comprising:
(a) culturing bacterial cells, which cells comprise nucleic acid encoding
phage lysozyme and nucleic acid encoding a protein that displays DNA-digesting
activity under the control of a signal sequence for secretion of said DNA-
digesting protein, wherein said nucleic acids are linked to a first promoter,
and nucleic acid encoding the heterologous polypeptide and a signal sequence
for secretion of the heterologous polypeptide, which nucleic acid encoding the
heterologous polypeptide is linked to a second promoter, wherein the second
promoter is inducible and the first promoter is either a promoter with low
basal expression or an inducible promoter, the culturing being under
conditions
whereby when an inducer is added, expression of the nucleic acid encoding the
phage lysozyme and DNA-digesting protein is induced after about 50t or more of
the heterologous polypeptide has accumulated, whereby the phage lysozyme
accumulates in a cytoplasmic compartment, whereby the DNA-digesting protein is
secreted to the periplasm, and whereby the cells are lysed to produce a broth
lysate; and
(b) recovering accumulated heterologous polypeptide from the broth
lysate.
In yet another aspect, the invention supplies a process for recovering
a heterologous polypeptide from bacterial cells in which it is produced
comprising:
(a) culturing the bacterial cells, which cells comprise nucleic acid
encoding phage lysozyme, gene t, and nucleic acid encoding a protein that
displays DNA-digesting activity, wherein these nucleic acids are linked to a
first promoter, and nucleic acid encoding the heterologous polypeptide, which
nucleic acid is linked to a second promoter, wherein the second promoter is
inducible and the first promoter is either a promoter with low basal
expression
or an inducible promoter, the culturing being under conditions whereby when an
inducer is added, expression of the nucleic acids encoding the phage lysozyme,
gene t, and DNA-digesting protein is induced after about 50t or more of the
heterologous polypeptide has accumulated, and under conditions whereby the
phage lysozyme accumulates in a cytoplasmic compartment, whereby the DNA-
-5-
CA 02528202 1999-10-21
digesting protein is secreted into the periplasm, and whereby the cells are
lysed to produce a broth lysate; and
(b) recovering accumulated heterologous polypeptide from the broth
lysate.
In a third aspect, the invention provides a process for recovering a
heterologous polypeptide from bacterial cells in which it is produced
comprising:
(a) culturing the bacterial cells, which cells comprise nucleic acid
encoding phage lysozyme, gene t, and nucleic acid encoding a protein that
displays DNA-digesting activity; wherein the nucleic acid encoding the phage
lysozyme and DNA-digesting protein is linked to a first promoter that is
inducible or with low basal expression, the gene t is linked to a second
inducible promoter, and the nucleic acid encoding the heterologous polypeptide
is linked to a third inducible promoter, under conditions whereby when an
inducer is added and all three promoters are inducible, expression of the
nucleic acids encoding the phage lysozyme, gene t, and DNA-digesting protein
is induced after about 50% or more of the heterologous polypeptide has
accumulated, with the third promoter being induced before the first promoter
and the second promoter induced after the first promoter, and whereby if the
phage lysozyme and DNA-digesting protein are linked to a promoter with low
basal expression, expression of the gene t is induced after about 50% or more
of the heterologous polypeptide has accumulated, and under conditions whereby
the phage lysozyme accumulates in a cytoplasmic compartment, whereby the DNA-
digesting protein is secreted into the periplasm, and whereby the cells are
lysed to produce a broth lysate; and
(b) recovering accumulated heterologous polypeptide from the broth
lysate.
Biochemical lysis or biochemically-assisted mechanical lysis is superior
to mechanical disruption for recovering heterologous polypeptide from
bacterial
cells. Coordinated expression of nucleic acid encoding phage lysozyme with
gene t and DNA-digesting protein, and nucleic acid encoding the heterologous
polypeptide of interest provides a highly effective method for releasing
insoluble or soluble polypeptide from the entanglement with the peptidoglycan
layer, as well as releasing product trapped in the cytoplasm. When the phage
lysozyme gene is cloned behind a tightly-controlled promoter, for example, the
pBAD promoter (also referred to as the ara promoter). cytoplasmic accumulation
of phage lysozyme may be induced by the addition of an inducer (such as
arabinose) at an appropriate time near the end of fermentation. By placing the
nucleic acid expression of heterologous polypeptide and lytic enzymes under
separate promoter control, one can independently regulate their production
during fermentation. Without a signal sequence, the accumulated phage lysozyme
is tightly locked up in the cytoplasmic compartment, and gene t functions to
release the phage lysozyme to degrade the peptidoglycan layer. Furthermore,
-6-
CA 02528202 1999-10-21
the optimal pH for T4-phage-lysozyme activity, which is a preferred
embodiment,
is about 7.3, which is about the neutral pH of most typical harvest broths.
The induction of the genes encoding the bacteriophage lysozyme, DNA-
digesting protein, and gene t after expression of the nucleic acid encoding
the
heterologous polypeptide results in a significant amount of insoluble or
soluble heterologous polypeptide recovered from the cytoplasm or periplasm of
bacteria. Besides product yield, the success of a recovery process is judged
by the ease of operation, the process flow, the turn-around time, as well as
the operation cost. The present invention alleviates several if not all these
bottlenecks encountered in the large-scale recovery process.
The processes herein also allow use of biochemical cell lysis at high
cell density and increased scale. At high density, excessive expression of T4-
lysozyme, gene t, and endA could have disastrous results, such as premature
cell lysis and reduction in heterologous polypeptide production. Further, it
would not be expected that induction at the end of a long fermentation process
and after substantial product accumulation would produce enough of the lytic
enzymes to be effective. The present processes do not pose problems at high
cell densities such as increased viscosity and excessive foaming during the
fermentation process. It is expected that the processes herein will enable the
attainment of high cell density, effective induction and action of the system,
and the processing of broth lysates derived from high-density cultures.
Brief Description of the Drawings
Figure 1 depicts a schematic diagram of how a polypeptide product is
disposed in the cytoplasm and in the periplasm, that is, it forms an
aggregate,
proteolyzed fragment, or folded soluble polypeptide.
Figure 2 depicts IGF-I aggregate recovery from the supernatant and pellet
by the typical isolation procedure involving mechanical cell disruption
followed by centrifugation, after three passes through the Gaulin homogenizer.
Figure 3 depicts a plasmid map of pS1130, an expression plasmid for
rhuMAb CD18 F(ab')2-leucine zipper precursor (herein also referred to as anti-
CD18 antibody fragment).
Figures 4A-4B show the sequence of the expression cassette of pS1130 (SEQ
ID NO:1 and NO:2).
Figure 5 shows plasmid construction of pJJ154 used to co-express T4-
lysozyme and endA (H. coil DNase).
Figure 6 shows a plasmid map of pLBIGF57 used to express IGF-I.
Figure =7 shows plasmid construction of pJJ155 used to express T4-
lysozyme, endA, and gene t, which construction is from pJJ154.
Figure 8 depicts a schematic of the two-plasmid system for co-expression
of T4-lysozyme, a preferred phage lysozyme, endA, a preferred DNA-digesting
protein, and gene t (pJJ155) with IGF-I-encoding nucleic acid in accordance
with an example of this invention.
-7-
CA 02528202 1999-10-21
Figures 9A-9E disclose photographs from phase-contrast microscopy of the
harvest broth and resuspended pellets from centrifugation of fermentation
broth
with and without co-expression of T4-lysozyme and endA and t-gene before and
after EDTA addition. Specifically, the photographs show the resuspended pellet
from centrifugation of control broth with no lytic enzyme co-expression before
and after EDTA addition respectively (Figs. 9A and 9B), the fermentation
harvest undiluted whole broth resulting from co-expression of IGF-I with the
three lytic enzymes, T4-lysozyme, endA, and t-gene (Fig. 9C), the resuspended
pellet from centrifugation of harvest broth with co-expression of IGF-I with
the three lytic enzymes and no EDTA addition (Fig. 9D), and the resuspended
pellet from centrifugation of harvest broth with the co-expression of IGF-I
with the three lytic enzymes and EDTA addition (Fig. 9E).
Figures 10A and lOB show, respectively, nucleic acid quantitation in the
supernatant and pellet by OD260 determination for IGF-I-expressing E. coli
with
co-expression of the three lytic enzymes versus control, and total protein in
the supernatant and pellet from IGF- I -expressing S. coli with co-expression
of
the three lytic enzymes versus no co-expression control.
Figure 11 shows IGF-I product recovery by centrifugation using three
lytic enzymes versus no-lysis control broth for various centrifugation speeds.
Figure 12 shows solids recovery during centrifugation using three lytic
enzymes co-expressed with IGF-I versus no-lysis control broth for various
centrifugation speeds.
Detailed Description of the Preferred Embodiments
A. Definitions
As used herein, "phage lysozyme' refers to a cytoplasmic enzyme that
facilitates lysis of phage-infected bacterial cells, thereby releasing
replicated phage particles. The lysozyme may be from any bacteriophage source,
including T7, T4, lambda, and mu bacteriophages. The preferred such lysozyme
herein is T4-lysozyme.
As used herein, "T4-lysozyme" or "E protein" refers to a cytoplasmic
muramidase that facilitates lysis of T4 phage-infected bacterial cells,
thereby
releasing replicated phage particles (Tsugita and Inouye, J. Mol. Biol., 37:
201-12 (1968); Tsugita and Inouye, J. Biol. Chem., 1: 391-97 (1968)). It is
encoded by gene e of T4 bacteriophage and hydrolyzes bonds between N-
acetylglucosamine and N-acetylmuramic acid residues in the rigid peptidoglycan
layer of the E. coli cell envelope. The enzyme is a single polypeptide chain
of a molecular weight of 18.3 kd. It is approximately 250-fold more active
than HEW-lysozyme against bacterial peptidoglycan (Matthews at al., J. Mol.
Biol., 147: 545-558 (1981)). The optimal pH for T4-lysozyme enzyme activity
is 7.3, versus 9 for HEW-lysozyme (The Worthington Manual; pp 219-221).
As used herein, "gene t' or "t gene" or "holin' refers to a lytic gene
of bacteriophage T4 that is required for lysis but does not appear to have
-8-
CA 02528202 1999-10-21
lysozyme activity. See also Molecular Genetics of Bacterioohaae T4, supra, p.
398-399.
The term "protein that displays DNA-digesting activity" or 'DNA-
digesting protein" refers to a protein that will digest DNA such as, for
example, mammalian or bacterial DNase. Preferably, the DNA-digesting protein
is human DNase or bacterial endA.
As used herein, the phrase "lytic enzymes" refers collectively to at
least phage lysozyme and DNA-digesting protein;. where applicable it also
refers
to a phage gene t gene product or equivalent in combination with phage
lysozyme
and DNA-digesting protein.
As used herein, the phrase "agent that disrupts the outer cell wall" of
bacteria refers to a molecule that increases permeability of the outer cell
wall of bacteria, such as chelating agents, e.g., EDTA, and zwitterions.
As used herein, the term "bacteria" refers to any bacterium that produces
proteins that are transported to the periplasmic space. Generally, the
bacteria, whether gram positive or gram negative, has phage lysozyme and
nuclease expression under control so that they are only expressed near the end
of the fermentation, a preferred embodiment, or expressed at a low level
during
fermentation. The nuclease is generally relatively stable when secreted to the
periplasm or medium. The term "non-temperature-sensitive bacteria" refers to
any bacterium that is not significantly sensitive to temperature changes. One
preferred embodiment herein is bacteria that are not temperature sensitive.
The most preferred bacteria herein are gram-negative bacteria.
As used herein, "a time sufficient to release the polypeptide contained
in the cytoplasm or periplasm" refers to an amount of time sufficient to allow
the lysozyme to digest the peptidoglycan to a sufficient degree to release the
cytoplasmic or periplasmic aggregate or soluble heterologous polypeptide.
As used herein, "signal sequence" or "signal polypeptide" refers to a
peptide that can be used to secrete the heterologous polypeptide or protein
that displays DNA-digesting activity into the periplasm of the bacteria. The
signals for the heterologous polypeptide or DNA-digesting protein may be
homologous to the bacteria, or they may be heterologous, including signals
native to the heterologous polypeptide or DNA-digesting protein being produced
in the bacteria.
The promoters of this invention may be "inducible" promoters, i.e.,
promoters that direct transcription at an increased or decreased rate upon
binding of a transcription factor.
As used herein, a promoter "with low basal expression" or a "low-basal-
expression promoter" is a promoter that is slightly leaky, i.e., it provides
a sufficiently low basal expression level so as not to affect cell growth or
product accumulation and provides a sufficiently low level of promotion not to
result in premature cell lysis.
-9-
CA 02528202 1999-10-21
"Transcription factors" as used herein include any factors that can bind
to a regulatory or control region of a promoter and thereby effect
transcription. The synthesis or the promoter-binding ability of a
transcription factor within the host cell can be controlled by exposing the
host to an "inducer" or removing a "repressor" from the host cell medium.
Accordingly, to regulate expression of an inducible promoter, an inducer is
added or a repressor removed from the growth medium of the host cell.
As used herein, the phrase "induce expression" means to increase the
amount of transcription from specific genes by exposure of the cells
containing
such genes to an effector or inducer.
An "inducer" is a chemical or physical agent which, when given to a
population of cells, will increase the amount of transcription from specific
genes. These are usually small molecules whose effects are specific to
particular operons or groups of genes, and can include sugars, alcohol, metal
ions, hormones, heat, cold, and the like. For example, isopropylthio-p-
galactoside (IPTG) and lactose are inducers of the tacll promoter, and L-
arabinose is a suitable inducer of the arabinose promoter.
A "repressor" is a factor that directly or indirectly leads to cessation
of promoter action or decreases promoter action. One example of a repressor
is phosphate. As the repressor phosphate is depleted from the medium, the
alkaline phosphatase (AP) promoter is induced.
As used herein, "polypeptide" or "polypeptide of interest" refers
generally to peptides and proteins having more than about ten amino acids. The
polypeptides are "heterologous," i.e., foreign to the host cell being
utilized,
such as a human protein produced by E. coil. The polypeptide may be produced
as an insoluble aggregate or as a soluble polypeptide in the periplasmic space
or cytoplasm.
Examples of mammalian polypeptides include molecules such as, e.g.,
renin, a growth hormone, including human growth hormone; bovine growth
hormone;
growth hormone releasing factor; parathyroid hormone; thyroid stimulating
hormone; lipoproteins; al-antitrypsin; insulin A-chain; insulin B-chain;
proinsulin; thrombopoietin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor
IX, tissue factor, and von Willebrands factor; anti-clotting factors such as
Protein C; atrial naturietic factor; lung surfactant; a plasminogen activator,
such as urokinase or human urine or tissue-type plasminogen activator (t-PA);
bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and
-beta; enkephalinase; a serum albumin such as human serum albumin; mullerian-
inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse
gonadotropin-associated peptide; a microbial protein, such as beta-lactamase;
DNase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors
for hormones or growth factors; integrin; protein A or D; rheumatoid factors;
a neurotrophic factor such as brain-derived neurotrophic factor (BDNF),
-10-
CA 02528202 1999-10-21
neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth
factor such as NGF-B; cardiotrophins (cardiac hypertrophy factor) such as
cardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF); fibroblast
growth factor such as aFGF and bFGF; epidermal growth factor (EGF);
transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-
ai, TGF-02, TGF-03, TGF-04, or TGF-(i5; insulin-like growth factor-I and -II
(IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor
binding proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;
erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic
protein (BMP); an interferon such as interferon-alpha,
-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and
G-CSF; interleukins (ILS), e.g., IL-1 to IL-l0; anti-HER-2 antibody;
superoxide
dismutase; T-cell receptors; surface membrane proteins; decay accelerating
factor; viral antigen such as, for example, a portion of the AIDS envelope;
transport proteins; homing receptors; addressins; regulatory proteins;
antibodies; and fragments of any of the above-listed polypeptides.
The preferred exogenous polypeptides of interest are mammalian
polypeptides, most preferably human polypeptides. Examples of such mammalian
polypeptides include t-PA, VEGF, gp120, anti-HER-2, anti-CD11a, anti-CD18,
DNase, IGF-I, IGF-II, brain IGF-I, growth hormone, relaxin chains, growth
hormone releasing factor, insulin chains or pro-insulin, urokinase,
immunotoxins, neurotrophins, and antigens. Particularly preferred mammalian
polypeptides include, e.g., IGF-I, DNase, or VEGF, most preferably IGF-I, if
the polypeptide is produced as an insoluble aggregate in the periplasm, and
anti-CD18 antibodies or fragments thereof such as anti-recombinant human CD18
Fab, Fab' and (Fab')2 fragments, if the polypeptide is produced in a soluble
form in the periplasm.
As used herein, "IGF-I" refers to insulin-like growth factor from any
species, including bovine, ovine, porcine, equine, and preferably human, in
native-sequence or in variant form and recombinantly produced. In a preferred
method, the IGF-I is cloned and its DNA expressed, e.g., by the process
described in EP 128,733 published December 19, 1984.
The expression "control sequences" refers to DNA sequences necessary for
the expression of an operably-linked coding sequence in a particular host
organism. The control sequences that are suitable for bacteria include a
promoter such as the alkaline phosphatase promoter, optionally an operator
sequence, and a ribosome-binding site.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For example, DNA for a
presequence or secretory leader is operably linked to DNA for a heterologous
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked to a
coding sequence if it affects the transcription of the sequence; or a ribosome
-11-
CA 02528202 2008-09-05
binding site is operably linked to a coding sequence if it is positioned so as
to
facilitate translation. Generally,"operably linked"means that the DNA
sequences
being linked are contiguous and, in the case of a secretory leader, contiguous
and
in reading frame. Linking is accomplished by ligation at convenient
restriction
sites. If such sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
As used herein, the expressions"cell,""cell line,"and"cell culture" are used
interchangeably and all such designations include progeny. Thus, the
words"transformants"and"transformed cells"include the primary subject cell and
cultures derived therefrom without regard for the number of transfers. It is
also
understood that all progeny may not be precisely identical in DNA content, due
to
deliberate or inadvertent mutations. Mutant progeny that have the same
function or
biological activity as screened for in the originally transformed cell are
included. Where distinct designations are intended, it will be clear from the
context.
B. Modes for Carrying Out the Invention
In various aspects, the invention provides processes for recovering a
heterologous
polypeptide, soluble or insoluble, from bacterial cells in which it is
produced.
In selected embodiments, the invention provides processes for recovering a
heterologous polypeptide from bacterial cells comprising:
(a) culturing the cells, which comprise a first nucleic acid encoding the
heterologous polypeptide which is linked to a first inducible promoter, a
second
nucleic acid encoding phage lysozyme, a third nucleic acid encoding a protein
that
displays DNA-digesting activity under the control of a signal sequence for
secretion of the DNA-digesting protein, and gene t, wherein either:
(i) said second and third nucleic acids and said gene t are
operatively linked to a second inducible promoter that is the same for all
three and wherein all three are linked on the same nucleic acid construct,
or
(ii) said second and third nucleic acids are operatively linked to a
second inducible promoter that is the same for both and wherein both are
linked on the same nucleic acid construct, and said gene t is operatively
linked to a third inducible promoter, or
(iii) said second and third nucleic acids are operatively linked to a
weak constitutive promoter or a promoter with a low basal level that does
not require the addition of an inducer to function as a promoter, and said
gene t is operably linked to a second inducible promoter;
wherein the first, second, and third inducible promoters are different from
each
other and respond to different inducers; and either:
(b) when the culturing is by (a)(i) above, adding an inducer specific for
induction of expression of the nucleic acid encoding the heterologous
polypeptide
from the first inducible promoter, and then adding an inducer specific for the
-12a-
CA 02528202 2008-09-05
second inducible promoter after accumulation of about 50% or more of the
maximum
accumulation of the heterologous polypeptide to be recovered; or
(c) when the culturing is by (a)(ii) above, adding an inducer specific for
induction of expression of the nucleic acid encoding the heterologous
polypeptide
from the first inducible promoter, then adding after accumulation of about 50%
or
more of the maximum accumulation of the heterologous polypeptide to be
recovered an
inducer specific for the second inducible promoter, and then adding an inducer
specific for the third inducible promoter; or
(d) when the culturing is by (a) (iii) above, adding an inducer specific
for induction of expression of the nucleic acid encoding the heterologous
polypeptide from the first inducible promoter, and then adding an inducer
specific
for the second inducible promoter after accumulation of about 50% or more of
the
maxim accumulation of the heterologous polypeptide to be recovered, wherein
the
cells are lysed and the lysing results in a broth lysate; and
(e) recovering accumulated heterologous polypeptide from the broth lysate
thus produced.
In selected embodiments, the heterologous polypeptide may be a mammalian
polypeptide, such as a human polypeptide, such as an insulin-like growth
factor-I
(IGF-I), DNase, vascular endothelial growth factor (VEGF), or anti-CD18
antibody or
fragment thereof. A signal sequence, which may be identified as a secretory
signal
sequence, may be used to secrete the heterologous polypeptide into the
periplasm of
the bacterial cells, such as a signal sequence that is a native sequence of
the
DNA-digesting protein. The DNA-digesting protein may for example be a
eukaryotic
DNase or bacterial endA. The phage lysozyme may for example be phage T4-
lysozyme.
The heterologous polypeptide may for example be soluble in the periplasmic
space,
and the recovery step may be carried out using an expanded bed absorption
process
or centrifugation.
In selected embodiments, before recovery, the broth lysate may be incubated
for a
time sufficient to release the heterologous polypeptide contained in the
cells.
The recovery process may for example include sedimenting refractile particles
containing insoluble heterologous polypeptide or collecting supernatant
containing
soluble heterologous polypeptide, depending on whether the heterologous
polypeptide
is soluble or insoluble. The recovery step may for example take place in the
presence of an agent that disrupts the outer cell wall of the bacterial cells,
such
as a chelating agent or zwitterion.
The bacterial cells may be Gram-negative cells, such as E. coli cells. In
selected
embodiments, one or more of the nucleic acids or the gene t, including the
promoter
therefor, may be integrated into the genome of the bacterial cells.
-12b-
CA 02528202 2008-09-05
In an alternative embodiment, the invention provides a process that involves,
in a
first step, culturing the bacterial cells, which cells comprise nucleic acid
encoding the lytic enzymes, wherein these nucleic acids are linked to a first
promoter, and nucleic acid encoding the heterologous polypeptide, which
nucleic
acid is linked to a second inducible promoter. In an alternative embodiment,
the
phage lysozyme and DNA- digesting protein are linked to a first inducible
promoter
or a promoter with low basal expression, the t-gene is linked to a second
inducible
promoter, and the heterologous polypeptide is linked to a third inducible
promoter.
The culturing takes place under conditions whereby expression of the nucleic
acids
encoding the lytic enzymes, when induced, commences after about 50% or more of
the
heterologous polypeptide has accumulated, and under conditions whereby the
phage
lysozyme accumulates in a cytoplasmic compartment and the DNA-digesting
protein is
secreted into the periplasmic compartment.
In the processes herein, induction of the promoters is preferred; however, the
processes also contemplate the use of a promoter for the phage lysozyme and
DNA-
digesting protein that is a promoter with low basal expression (slightly
leaky),
wherein no induction is carried out. This type of promoter has a leakiness
that is
low enough not to result in premature cell lysis and results in a sufficiently
low
basal expression level so as not to affect cell growth or product
accumulation.
In a second step, the accumulated heterologous polypeptide is recovered from
the
bacterial cells. An agent that increases permeability of the outer cell wall
of the
bacterial cells may be added, as described in detail below, before the
recovery
step is carried out. The need to disrupt cells mechanically
-12c-
CA 02528202 1999-10-21
to release the phage lysozyme is either reduced or is completely avoided. in
a preferred embodiment, after lytic enzyme expression the cells are incubated
for a time sufficient to release the heterologous polypeptide contained in the
cytoplasm or periplasm.
while the processes can apply to the recovery of insoluble aggregates
such as IGF-I, VEGF, and DNase by sedimentation of product, they are also
applicable to heterologous polypeptides that are soluble in the cytoplasm or
periplasm, such as, for example, anti-CD18 antibody fragment. Advantages for
recovery of soluble heterologous polypeptides by biochemical cell lysis
include
avoiding or reducing the need for mechanical lysis, thereby avoiding loss of
heat-labile proteins, and obtaining a low and consistent fluid viscosity
compatible with downstream recovery processes such as expanded bed absorption
technology and centrifugation.
Expanded bed absorption (EBA) chromatography, described, for example, in
"Expanded Bed Adsorption: Principles and Methods', Pharmacia Biotech, ISBN
91-630-5519-8), is useful for the initial recovery of target proteins from
crude feed-stock or cell culture. The process steps of clarification,
concentration, and initial purification can be combined into one unit
operation, providing increased process economy due to a decreased number of
process steps, increased yield, shorter overall process time, reduced labor
coat, and reduced cost. In EBA chromatography an adsdrbent is expanded and
equilibrated by applying an upward liquid flow to the column. A stable
fluidized bed is formed when the adsorbent particles are suspended in
equilibrium due to the balance between particle sedimentation velocity and
upward liquid flow velocity. Crude cell mixture or broth lysate is applied to
the expanded bed with an upward flow. Target proteins are bound to the
adsorbent while cell debris and other contaminants pass through unhindered.
weakly bound material is washed from the expanded bed using upward flow of a
wash buffer. Flow is then stopped and the adsorbent is allowed to settle in
the column. The column adaptor is then lowered to the surface of the
sedimented bed. Flow is reversed and the captured proteins are eluted from the
sedimented bed using an appropriate buffer. The eluate contains the target
protein in a reduced elution pool volume, partially purified in preparation
for
packed bed chromatography (Pharmacia Biotech, supra). EBA, wherein the whole
cell lysate containing soluble product is pumped up through the column and the
protein is absorbed onto a resin (fluidized bed) and the cell debris flows
through, utilizes only one chromatography step, thereby saving a step.
In another embodiment, the invention provides a process for recovering
soluble heterologous polypeptide from the cytoplasm or periplasm of bacterial
cells. This process involves culturing bacterial cells, which cells comprise
nucleic acid encoding phage lysozyme and nucleic acid encoding a DNA-digesting
protein that displays DNA-digesting activity under the control of a signal
sequence for secretion of said DNA-digesting protein. In this process, the
-13-
CA 02528202 1999-10-21
nucleic acids are linked to a first promoter, and nucleic acid encoding the
heterologous polypeptide and a signal sequence for secretion of the
heterologous polypeptide, which nucleic acid encoding the heterologous
polypeptide is linked to a second inducible promoter. This culturing takes
place under conditions whereby over-expression of the nucleic acid encoding
the
phage lysozyme and DNA-digesting protein is weakly and constitutively promoted
or, if induced, commences after about 50% or more of the heterologous
polypeptide has accumulated, and under conditions whereby the heterologous
polypeptide and DNA-digesting protein are secreted into the periplasm of the
bacteria and the phage lysozyme accumulates in a cytoplasmic compartment.
In a second step, the resulting heterologous polypeptide is recovered
from the broth lysate.
In a third embodiment, the invention provides a process for recovering
a heterologous polypeptide from bacterial cells in which three different
promoters are used. Specifically, in the first step, the bacterial cells are
cultured, where the cells comprise nucleic acid encoding phage lysozyme, gene
t, and nucleic acid encoding a protein that displays DNA-digesting activity,
as well as nucleic acid encoding the heterologous polypeptide. The nucleic
acid encoding the lysozyme and DNA-digesting protein is linked to a first
promoter that is inducible or with low basal expression, the gene t is linked
to a second inducible promoter, and the nucleic acid encoding the heterologous
polypeptide is linked to a third inducible promoter.
The culturing is carried out under conditions whereby when an inducer is
added and all three promoters are inducible, expression of the nucleic acids
encoding the phage lysozyme, gene t, and DNA-digesting protein is induced
after
about 50% or more of the heterologous polypeptide has accumulated, with the
third promoter being induced before the first promoter, and the second
promoter
induced last, and whereby if the phage lysozyme and DNA-digesting protein are
linked to a promoter with low basal expression, expression of the gene t is
induced after about 50% or more of the heterologous polypeptide has
accumulated. Culturing is also carried out under conditions whereby the phage
lysozyme accumulates in a cytoplasmic compartment, whereby the DNA-digesting
protein is secreted to and accumulates in the periplasm, and whereby the cells
are lysed to produce a broth lysate.
In a second step, accumulated heterologous polypeptide is recovered from
the broth lysate.
In the above processes, while the signal sequence for the DNA-digesting
protein may be any sequence, preferably, it is a native sequence of the DNA-
digesting protein. Also, in a preferred embodiment, the DNA-digesting protein
is DNase or bacterial, e.g., S. coli endA product.
In a preferred embodiment, the culturing step takes place under
conditions of high cell density, that is, generally at a cell density of about
15 to 150 g dry weight/liter, preferably at least about 40, more preferably
-14-
CA 02528202 1999-10-21
about 40-150, most preferably about 40 to 100. In optical density, 120 OD550
(Assn) is about 50 g dry wt./liter. In addition, the culturing can be
accomplished using any scale, even very large scales of 100,000 liters.
Preferably, the scale is about 100 liters or greater, more preferably at least
about 500 liters, and most preferably from about 500 liters to 100,000 liters.
The nucleic acids encoding the lytic enzymes are linked to one promoter,
i.e., put in tandem, as by placing a linker between the nucleic acids. The
promoter for the heterologous polypeptide expression is different from that
used for the lytic enzymes, such that one is induced before the other. while
the promoters may be any suitable promoters for this purpose, preferably, the
promoters for the lytic enzymes with or without gene t and heterologous
polypeptide are, respectively, arabinose promoter and alkaline phosphatase
promoter.
The promoters for the heterologous polypeptide and for the lytic enzymes
for all three processes herein must be different, such that the nucleic-acid-
encoded heterologous polypeptide expression is induced before expression of
nucleic-acid-encoded lytic enzymes or at a much higher level, when the
promoters are inducible. While the promoters may be any suitable promoters for
this purpose, preferably, the promoters for the phage lysozyme and
heterologous
polypeptide are, respectively, arabinose promoter and alkaline phosphatase
promoter. Alternatively, the compartmentalization of the phage lysozyme and
DNA-digesting protein may allow for the use of a promoter with low basal
expression for expression of the nucleic acid encoding phage lysozyme and DNA-
digesting protein. If a promoter with low basal expression is employed, such
as arabinose as opposed to tac or trp promoter, then an active step of
induction is not required.
The induction of expression of the nucleic acid encoding the lytic
enzymes is preferably carried out by adding an inducer to the culture medium.
While, in this respect, the inducers for the promoters may be added in any
amount, preferably if the inducer is arabinose, it is added in an amount of
about 0-1% by weight, and if inducer is added, 0.1-1% by weight.
In the processes described above, typically the expression elements are
introduced into the cells by transformation therein, but they may also be
integrated into the genome or chromosome of the cells along with their
promoter
regions. This applies to any of the lytic enzymes or the heterologous
polypeptide gene. The bacterial cells may be transformed with one or more
expression vectors containing the nucleic acid encoding the lytic enzymes, and
the nucleic acid encoding the heterologous polypeptide. In one such
embodiment, the bacterial cells are transformed with two vectors respectively
containing the nucleic acid encoding the lytic enzymes and the nucleic acid
encoding the heterologous polypeptide. In another embodiment, the nucleic acid
encoding the lytic enzymes and the nucleic acid encoding the heterologous
polypeptide are contained on one vector with which the bacterial cells are
-15-
CA 02528202 1999-10-21
transformed. In another specific embodiment that may be preferred, the phage
lysozyme and DNA-digesting enzyme are carried on a plasmid to ensure high copy
number and the gene t is integrated into the host chromosome to down-regulate
expression and prevent premature cell lysis to avoid leakiness.
in the first step of the above processes, the heterologous nucleic acid
(e.g., cDNA or genomic DNA) is suitably inserted into a replicable vector for
expression in the bacterium under the control of a suitable promoter for
bacteria. Many vectors are available for this purpose, and selection of the
appropriate vector will depend mainly on the size of the nucleic acid to be
inserted into the vector and the particular host cell to be transformed with
the vector. Each vector contains various components depending on its function
(amplification of DNA or expression of DNA) and the particular host cell with
which it is compatible. The vector components for bacterial transformation may
include a signal sequence for the heterologous polypeptide and will include a
signal sequence for the DNA-digesting protein and will also include an
inducible promoter for the heterologous polypeptide and gene t and an
inducible
promoter or a non-inducible one with low basal expression for the other lytic
enzymes. They also generally include an origin of replication and one or more
marker genes.
In general, plasmid vectors containing replicon and control sequences
that are derived from species compatible with the host cell are used in
connection with bacterial hosts. The vector ordinarily carries a replication
site, as well as marking sequences that are capable of providing phenotypic
selection in transformed cells. For example, E. coli is typically transformed
using pBR322, a plasmid derived from an E. coli species. See, e.g., Bolivar
et al., Gene, ?: 95 (1977). pBR322 contains genes conferring ampicillin and
tetracycline resistance and thus provides an easy means for identifying
transformed cells. The pBR322 plasmid, or other microbial plasmid or phage,
also generally contains, or is modified to contain, promoters that can be used
by the bacterial organism for expression of the selectable marker genes.
If the heterologous polypeptide is to be secreted, the DNA encoding the
heterologous polypeptide of interest herein contains a signal sequence, such
as one at the N-terminus of the mature heterologous polypeptide. In general,
the signal sequence may be a component of the vector, or it may be a part of
the heterologous polypeptide DNA that is inserted into the vector. The
heterologous signal sequence selected should be one that is recognized and
processed (i.e., cleaved by a signal peptidase) by the host cell. For
bacterial host cells that do not recognize and process the native heterologous
polypeptide signal sequence, the signal sequence is substituted by a bacterial
signal sequence selected, for example, from the group consisting of the
alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II
leaders.
-16-
CA 02528202 1999-10-21
Expression vectors contain a nucleic acid sequence that enables the
vector to replicate in one or more selected host cells. Such sequences are
well known for a variety of bacteria. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria.
S Expression vectors also generally contain a selection gene, also termed
a selectable marker. This gene encodes a protein necessary for the survival
or growth of transformed host cells grown in a selective culture medium. Host
cells not transformed with the vector containing the selection gene will not
survive in the culture medium. Typical selection genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients not available from complex
media, e.g., the gene encoding D-alanine racemase for Bacilli. One example of
a selection scheme utilizes a drug to arrest growth of a host cell. Those
cells that are successfully transformed with a heterologous gene produce a
protein conferring drug resistance and thus survive the selection regimen.
The expression vector for producing a heterologous polypeptide also
contains an inducible promoter that is recognized by the host bacterial
organism and is operably linked to the nucleic acid encoding the heterologous
polypeptide of interest. It also contains a separate inducible or low-basal-
expression promoter operably linked to the nucleic acid encoding the lytic
enzymes. Inducible promoters suitable for use with bacterial hosts include the
(i-lactamase and lactose promoter systems (Chang at al., Nature, 275: 615
(1978); Goeddel et al., Nature, 281: 544 (1979)), the arabinose promoter
system, including the araBAD promoter (Guzman et al., J. Bacteriol., 174: 7716-
7728 (1992); Guzman at al., J. Bacteriol., 177: 4121-4130 (1995); Siegele and
Hu, Proc. Natl. Acad. Sci. USA, li: 8168-8172 (1997)), the rhamnose promoter
(Haldimann et al., J. Bacteriol., 180: 1277-1286 (1998)), the alkaline
phosphatase promoter, a tryptophan (tip) promoter system (Goeddel, Nucleic
Acids Res., $: 4057 (1980) and EP 36,776), the PLteto-1 and Plac/ara-1
promoters
(Lutz and Bujard, Nucleic Acids Rea., 21: 1203-1210 (1997)), and hybrid
promoters such as the tac promoter. deBoer at al., Proc. Natl. Acad. Sci. USA,
e0: 21-25 (1983). However, other known bacterial inducible promoters and low-
basal-expression promoters are suitable. Their nucleotide sequences have been
published, thereby enabling a skilled worker operably to ligate them to DNA
encoding the heterologous polypeptide of interest or to the nucleic acids
encoding the lytic enzymes (Siebenlist et al., Cell, 20: 269 (1980)) using
linkers or adaptors to supply any required restriction sites. If a strong and
highly leaky promoter, such as the trp promoter, is used, it is generally used
only for expression of the nucleic acid encoding the heterologous polypeptide
and not for lytic -enzyme -encoding nucleic acid. The tac and PL promoters
could
be used for either, but not both, the heterologous polypeptide and the lytic
-17-
CA 02528202 1999-10-21
enzymes, but are not preferred. Preferred are the alkaline phosphatase
promoter for the product and the arabinose promoter for the lytic enzymes.
Promoters for use in bacterial systems also generally contain a Shine-
Dalgarno (S.D.) sequence operably linked to the DNA encoding the heterologous
polypeptide of interest. The promoter can be removed from the bacterial source
DNA by restriction enzyme digestion and inserted into the vector containing
the
desired DNA. The phoA promoter can be removed from the bacterial- source DNA
by restriction enzyme digestion and inserted into the vector containing the
desired DNA.
Construction of suitable vectors containing one or more of the above-
listed components employs standard ligation techniques. Isolated plasmids or
DNA fragments are cleaved, tailored, and re-ligated in the form desired to
generate the plasmids required.
For analysis to confirm correct sequences in plasmids constructed, the
ligation mixtures are used to transform E. coli K12 strain 294 (ATCC 31,446)
or other strains, and successful transformants are selected by ampicillin or
tetracycline resistance where appropriate. Plasmids from the transformants are
prepared, analyzed by restriction endonuclease digestion, and/or sequenced by
the method of Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977)
or Messing et al., Nucleic Acids Res., 9: 309 (1981), or by the method of
Maxam
et al., Methods in Enzymology, 65: 499 (1980).
Suitable bacteria for this purpose include archaebacteria and eubacteria,
especially eubacteria, more preferably Gram-negative bacteria, and most
preferably Enterobacteriaceae. Examples of useful bacteria include
Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas,
Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla,
and Paracoccus. Suitable E. coli hosts include E. coli W3110 (ATCC 27,325),
E. coli 294 (ATCC 31,446), E. coli B, and E. coli X1776 (ATCC 31,537). These
examples are illustrative rather than limiting. Mutant cells of any of the
above-mentioned bacteria may also be employed. it is, of course, necessary to
select the appropriate bacteria taking into consideration replicability of the
replicon in the cells of a bacterium. For example, E. coli, Serratia, or
Salmonella species can be suitably used as the host when well-known plasmids
such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
E. coli strain W3110 is a preferred host because it is a common host
strain for recombinant DNA product fermentations. Preferably, the host cell
should secrete minimal amounts of proteolytic enzymes. For example, strain
W3110 may be modified to effect a genetic mutation in the genes encoding
proteins, with examples of such hosts including E. coli W3110 strain 1A2,
which
has the complete genotype tonAA (also known as AfhuA); E. coli W3110 strain
9E4, which has the complete genotype tonAM ptr3; E. coli W3110 strain 27C7
(ATCC 55,244), which has the complete genotype tonAd ptr3 phoA4E15 d(argF-
-18-
CA 02528202 1999-10-21
lac)169 ompTA degP4lkanr; E. coli W31l0 strain 37D6, which has the complete
genotype tonAe ptr3 phoAAE15 6(argF-lac)169 omgTA degP4lkanr rbs7d ilvG; E.
coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; E. coli W3110 strain 33D3, which has the complete
genotype tonA ptr3 laclq LacL8 ompT degP kanr; E. coli W3110 strain 36F8,
which
has the complete genotype tonA phoA A(argF-lac) ptr3 degP kanR ilvG+, and is
temperature resistant at 37 C; E. coli W3110 strain 45F8, which has the
complete genotype fhuA (tonA) d(argF-lac) ptr3 degP41(kanS) d omp A(nmpc-fepE)
iivG+ phoA+ phoS*(T1OY); E. coli W3110 strain 33B8, which has the complete
genotype tonA phoA d(argF-lac) 189 deoC degP I1vG+(kanS); E. coil W3110 strain
43E7, which has the complete genotype fhuA (tonA) d (argF-lac) ptr3 degP41
(kanS)
dompTA(nmpc-fepE) ilvG+ phcA+; and an E. coli strain having the mutant
periplasmic protease(s) disclosed in U.S. Pat. No. 4,946,783 issued August 7,
1990.
Host cells are transformed with the above-described expression vectors
of this invention and cultured in conventional nutrient media modified as
appropriate for inducing the various promoters if induction is carried out.
Transformation means introducing DNA into an organism so that the DNA is
replicable, either as an extrachromosomal element or as chromosomal
integration. Depending on the host cell used, transformation is done using
standard techniques appropriate to such cells. The calcium treatment employing
calcium chloride, as described in section 1.82 of Sambrook et al., Molecular
Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
1989), is generally used for bacterial cells that contain substantial cell-
wall
barriers. Another method for transformation employs polyethylene glycol/DMSO,
as described in Chung and Miller, Nucleic Acids Res., lsi: 3580 (1988). Yet
another method is the use of the technique termed electroporation.
Bacterial cells used to produce the heterologous polypeptide of interest
described in this invention are cultured in suitable media in which the
promoters can be induced as described generally, e.g., in Sambrook et al.,
supra.
Any other necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate concentrations,
introduced alone or as a mixture with another supplement or medium such as a
complex nitrogen source. The pH of the medium may be any pH from about 5-9,
depending mainly on the host organism.
For induction, typically the cells are cultured until a certain optical
density is achieved, e.g., a AS50 of about 80-100, at which point induction is
initiated (e.g., by addition of an inducer, by depletion of a repressor,
suppressor, or medium component, etc.). to induce expression of the gene
encoding the heterologous polypeptide. When about 50% or more of the
heterologous polypeptide has accumulated (as determined, e.g., by the optical
density reaching a target amount observed in the past to correlate with the
-19-
CA 02528202 1999-10-21
desired heterologous polypeptide accumulation, e.g., a Asso of about 120-140),
induction of the promoter is effected for the lysis enzymes. The induction
typically takes place at a point in time post-inoculation about 75-90%,
preferably about 80-90%, of the total fermentation process time, as determined
from prior experience and assays. For example, induction of the promoter may
take place at from about 30 hours, preferably 32 hours, up to about 36 hours
post-inoculation of a 40-hour fermentation process.
Gene expression may be measured in a sample directly, for example, by
conventional northern blotting to quantitate the transcription of mRNA
(Thomas,
Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)). Various labels may be
employed, most commonly radioisotopes, particularly 32P. However, other
techniques may also be employed, such as using biotin-modified nucleotides for
introduction into a polynucleotide. The biotin then serves as the site for
binding to avidin or antibodies, which may be labeled with a wide variety of
labels, such as radionuclides, fluorescers, enzymes, or the like.
For accumulation of an expressed gene product, the host cell is cultured
under conditions sufficient for accumulation of the gene product. Such
conditions include, e.g., temperature, nutrient, and cell-density conditions
that permit protein expression and accumulation by the cell. Moreover, such
conditions are those under which the cell can perform basic cellular functions
of transcription,, translation, and passage of proteins from one cellular
compartment to another for the secreted proteins, as are known to those
skilled
in the art.
After product accumulation, when the cells have been lysed by the lytic
enzymes expressed, optionally before product recovery the broth lysate is
incubated for a period of time sufficient to release the heterologous
polypeptide contained in the cells. This period of time will depend, for
example, on the type of heterologous polypeptide being recovered and the
temperature involved, but preferably will range from about 1 to 24 hours, more
preferably 2 to 24 hours, and most preferably 2 to 3 hours. If there is
overdigestion with the enzyme, the improvement in recovery of product will not
be as great.
In the second step of this invention, the heterologous polypeptide, as
a soluble or insoluble product released from the cellular matrix, is recovered
from the lysate, in a manner that minimizes co-recovery of cellular debris
with
the product. The recovery may be done by any means, but preferably comprises
sedimenting refractile particles containing the heterologous polypeptide or
collecting supernatant containing soluble product. An example of sedimentation
is centrifugation. In this case, the recovery preferably takes place, before
EBA or sedimentation, in the presence of an agent that disrupts the outer cell
wall to increase permeability and allows more solids to be recovered. Examples
of such agents include a chelating agent such as EDTA or a zwitterion such as,
for example, a dipolar ionic detergent such as ZWITTERGENT 316i' detergent.
See
-20-
CA 02528202 1999-10-21
Stabel et al., supra. Most preferably, the recovery takes place in the
presence of EDTA. Another technique for the recovery of soluble product is
EBA, as described above.
If centrifugation is used for recovery, the relative centrifugal force
5= (RCF) is an important factor. The RCF is adjusted to minimize co-
sedimentation
of cellular debris with the refractile particles released from the cell wall
at lysis. The specific RCF used for this purpose will vary with, for example,
the type of product to be recovered, but preferably is at least about 3000 x
g, more preferably about 3500-6000 x g, and most preferably about 4000-6000 x
g. For the case with t-gene co-expression, about 6000 rpm provides as good a
recovery of refractile particles from lysed broth as for intact cells.
The duration of centrifugation will depend on several factors. The
sedimentation rate will depend upon, e.g., the size, shape, and density of the
refractile particle and the density and viscosity of the fluid. The
sedimentation time for solids will depend, e.g., on the sedimentation distance
and rate. It is reasonable to expect that the continuous disc-stack
centrifuges would work well for the recovery of the released heterologous
polypeptide aggregates or for the removal of cellular debris at large scale,
since these centrifuges can process at high fluid velocities because of their
relatively large centrifugal force and the relatively small sedimentation
distance.
The heterologous polypeptide captured in the initial recovery step may
then be further purified from the contaminating protein. In a preferred
embodiment, the aggregated heterologous polypeptide is isolated, followed by
a simultaneous solubilization and refolding of the polypeptide, as disclosed
in U.S. Pat. No. 5,288,931. Alternatively, the soluble product is recovered
by standard techniques.
The following procedures are exemplary of suitable purification
procedures for the soluble heterologous polypeptide released from the
periplasm
or the cytoplasm: fractionation on immunoaffinity or ion-exchange columns;
ethanol precipitation; reverse-phase HPLC; chromatography on silica or on a
cation-exchange resin such as DEAF; chromatofocusing; SDS-PAGE; ammonium
sulfate precipitation; and gel filtration using, for example, SEPHADEX= G-75.
The following examples are offered by way of illustration and not by way
of limitation.
EXAMPLE I
Co-expression of Lytic Enzymes with Soluble Product
A. Co-expression with T4-lvsozvme and endA endonuclease:
Background:
rhuMAb CD18 is a recombinant F(ab')2 antibody fragment with two 214-
residue light chains and two 241-residue heavy chains. It binds to the MAC-1
(CDllb/CD18) receptor, effectively blocking the binding of neutrophils to the
-21-
CA 02528202 1999-10-21
endothelium. In the fermentation process described below, rhuMab CD18 is
produced as a F(ab')2-leucine zipper precursor in E. coli and secreted into
the
periplasm. The desired recovery process targets the soluble fraction of the
accumulated product and depends on the F(ab')2-leucine zipper being released
from the periplasm for initial capture.
T4-lysozyme co-expression was introduced to weaken the peptidoglycan
sacculus, and the over-expression of endA protein was introduced to degrade
genomic DNA released from the cells under conditions such that the cells are
permeabilized or lysed.
In E. coli, the gene endA encodes for an endonuclease normally secreted
to the periplasm that cleaves DNA into ol igodeoxyribonucleot idea in an
endonucleolytic manner. It has been suggested that endA is relatively weakly
expressed by E. coli (Wackernagel et al., supra). However, one could
over-express the endonuclease with the use of a compatible plasmid. By
inserting the endA gene with its signal sequence behind the ara-T4-lysozyme
cassette in the compatible plasmid pJJ153, now pJJ154, expression of both
T4-lysozyme and endonuclease will be induced upon addition of arabinose. While
T4-lysozyme is locked inside the cytoplasm, endonuclease is secreted into the
periplasmic space and kept away from the genomic DNA located in the cytoplasm
during the fermentation process. An effective enzymatic degradation of DNA
upon cell lysis is expected to reduce or even eliminate multiple passes
through
the mechanical disruption device, an operation often needed for both cell
disruption and viscosity reduction. Success in doing so would bring
significant time and cost reduction to the recovery process.
Materials and Methods:
pS1130 Plasmid Construction: Plasmid pS1130 (Fig. 3) was constructed to direct
the production of the rhuMAb CD18 F(ab')2-leucine zipper precursor in E. coli.
It is based on the well-characterized plasmid pBR322 with a 2138-bp expression
cassette (Fig. 4; SEQ ID NO:1 and NO:2) inserted into the EcoRI restriction
site. The plasmid encodes for resistance to both tetracycline and beta-lactam
antibiotics. The expression cassette contains a single copy of each gene
linked in tandem. Transcription of each gene into a single dicistronic mRNA
is directed by the E. coli phoA promoter (Chang et al., Gene, 44: 121-125
(1986)) and ends at the phage lambda to terminator (Scholtissek and Grosse,
Nuc. Acids Res., 15: 3185 (1987)). Translation initiation signals for each
chain are provided by E. coli STII (heat-stable enterotoxin) (Picken et al.,
Infection and Immunity, 42: 269-275 (1983)) Shine-Dalgarno sequences.
rhuMAb CD18 was created by humanization of the murine monoclonal antibody
muMAb H52 (Hildreth and August, J. Immunology, 134: 3272-3280 (1985)) using a
process previously described for other antibodies (Carter et al., Proc. Natl.
Acad. Sci. USA, 89: 4285-4289 (1992); Shalaby et al., J. Exp. Med., 175: 217-
225 (1992); Presta at al., J. Immunol., 151: 2623-2632 (1993)). Briefly, cDNAs
encoding the muMAb H52 variable light (VL) and variable heavy (Vg) chain
domains
-22-
CA 02528202 1999-10-21
were isolated using RT-PCR from a hybridoma cell line licensed from Hildreth
(Hildreth and August, supra). The complementarity-determining regions (CDRs)
of the muMAb H52 were transplanted into the human antibody framework gene
encoding huMAb UCHT1-1 (Shalaby et al., supra) by site-directed mutagenesis.
Non-CDR, murine framework residues that might influence the affinity of the
humanized antibody for its target, human CD18, were identified using molecular
modeling. These residues were altered by site-directed mutagenesis and their
influence on CD18 binding was tested. Those framework residues that
significantly improved affinity were incorporated into the humanized antibody.
The expression vector encoding the final humanized version of muMAb H52 in an
Fab' format was named pH52-10.0 and it is a derivative of pAK19 (Carter et
al.,
Bio/Technology, 10: 163-167 (1992)).
Plasmid pS1130 differs from pH52-10.0 in the heavy-chain coding region.
The C-terminus of the heavy chain was extended from CysProPro to the natural
hinge sequence CysProProCysProAlaProLeuLeuGlyGly (SEQ ID NO:3) and then fused
to the 33-residue leucine zipper domain of the yeast transcription factor
GCN4.
As described above, the leucine zipper domains dimerize to bring two Fab'
molecules together and drive F(ab')2 complex formation. The two cysteine
residues in the heavy-chain hinge region then disulfide bond to those from an
adjacent Fab' to form a covalently-linked F(ab')2.
Plasmid pS1130 was constructed in a multi-step process outlined below:
First, a filamentous phage (f l) origin of replication was introduced into
pH52-10.0 to create plasmid pSO858. Plasmid pSO858 was constructed by ligating
the 1977-bp Hindlll-Hindlll fragment, containing the Fab' expression cassette
of pH52-10.0, with the 4870-bp HindIII-Hindiil fragment of plasmid pSOl91
(referred to as phGHr (1-238) in Fuh et al., J. Biol. Chem., 265: 3111-3115
(1990)).
Second, oligonucleotide-directed mutagenesis was performed on pSO858 to
create plasmid zpil#6. The heavy-chain coding region was extended to include
the 2-hinge cysteine residues and pepsin cleavage site:
(CysProProCysProAlaProLeuLeuGlyGly; SEQ ID NO:3). A NotI restriction site was
also introduced. The sequence of the introduced DNA was confirmed by DNA
sequencing.
Third, a DNA fragment encoding the GCN4 leucine zipper domain flanked by
=NotI and SphI restriction sites was generated (W098/37200 published August
27,
1998). This 107-bp NotI-SphI DNA fragment was subsequently cloned into
similarly-cut zipl#6 to create plasmid pSllll.
Fourth, DNA sequencing of the GCN4 leucine zipper fragment revealed an
error in the coding sequence. This error was corrected by oligonucleotide-
directed mutagenesis and confirmed by DNA sequencing. The resulting correct
plasmid was named pS1117.
The final step in the construction of pS1130 was to restore the
tetracycline-resistance gene and remove the fl origin from pS1117. This was
-23-
CA 02528202 1999-10-21
accomplished by ligating the 2884-bp PstI-SphI fragment of pS1117 containing
the Fab'-zipper expression cassette with the 3615-bp PstI-SphI fragment of
pH52-10Ø
1>JJ154 Plasmid Construction: Plasmid pJJ154 (Fig. 5) is a pACYC177 derivative
that is compatible with pBR322 vectors. To construct pJJ154, pJJ153 as
described below was digested with Mlul and the vector fragment was ligated
with
PCR-amplified endA gene designed to encode Mlul ends. The correct orientation
of the plasmid was screened for by restriction digest to produce pJJ154.
The construction of pJJ153 (a pACYC177 derivative that is compatible with
pBR322 vectors) is shown in Figure 5. The ClaI/AlwNI fragment from pBR322 was
inserted into ClaI/AlwNI-digested pBAD18 (Guzman et al., supra) to produce
pJJ70. One round of site-directed mutagenesis was then performed, changing
HindIII to Stul to obtain pJJ75. A second round of site-directed mutagenesis
was done to change MluI to SacII, to produce pJJ76. Then XbaI/HindIII
fragments from pJJ76 and from pBKIGF2B were ligated, and XbaI/HindIII
fragments
from this ligation product and from a T4 lysozyme/tac plasmid were ligated to
produce pT4LysAra. Then BamHI (filled in)/Scal-digested pACYC177 was ligated
with Clal/HindIII (both ends filled in)-digested pT4LysAra to produce pJJ153.
The maps for pACYC177, pT4LysAra, and pJJ153 are shown in Fig. 5.
Bacterial Strains and Growth Conditions: Strain 33B8 (E. coli W3110 tonA phoA
d(argF-lac) 189 deoC degP ilvG+(kanS)) was used as the production host for the
co-expression of T4-lysozyme and DNA-digesting protein from plasmid pJJ154 and
the expression of rhuMAb CD18 F(ab')2-leucine zipper from plasmid pS1130.
Competent cells of 33B8 were co-transformed with pJJ154 and pS1130 using
standard procedures. Transformants were picked from LB plates containing 20
ug/ml tetracycline and 50 ug/ml kanamycin (LB+Tet20+Kan50), streak-purified,
and grown in LB broth with 20 ug/ml tetracycline and 50 ug/ml kanamycin in a
37 C or 30 C shaker/incubator before being stored in DMSO at -80 C.
For control runs, the host 33B8 was transformed with pS1130 and pJJ96
(analogous to pJJ154 except no nucleic acids encoding the T4-lysozyme and endA
product were inserted into the vector) and isolated from similar selective
medium.
rhuMAb CD18 F(ab'),-Leucine Zipper Fermentation Process: A shake flask
inoculum was prepared by inoculating sterile medium using a freshly thawed
stock culture vial. Appropriate antibiotics were included in the medium to
provide selective pressure to ensure retention of the plasmid. The shake flask
medium composition is given in Table 1. Shake flasks were incubated with
shaking at about 30 C (28 C-32 C) for 14-18 hours. This culture was then used
to inoculate the production fermentation vessel. The inoculation volume was
between O.l% and 10% of the initial volume of medium.
-24-
CA 02528202 1999-10-21
The production of the F(ab')2-zipper precursor of rhuMAb CD18 was carried
out in the production medium given in Table 2. The fermentation process was
carried out at about 30 C (28-32 C) and about pH 7.0 (6.5-7.9). The aeration
rate and the agitation rate were set to provide adequate transfer of oxygen to
the culture. Production of the F(ab')z_zipper precursor of rhuMAb CD18
occurred
when the phosphate in the medium was depleted, typically 36-60 hours after
inoculation.
Table 1
Shake Flask medium composition
Ingredient Quantity/Liter
Tetracycline 4-20 mg
Tryptone 8-12 g
Yeast extract 4-6 g
Sodium chloride 8-12 g
Sodium phosphate, added as pH7 4-6 mmol
solution
Table 2
Production Medium composition
Ingredient Quantity/Liter
Tetracycline 4-20 mg
Glucose' 10-250 g
Ammonium sulfate' 2-8 g
Sodium phosphate, monobasic, 1-5 g
dihydrate'
Potassium phosphate, dibasic' 1-5 g
Potassium phosphate, monobasica 0.5-5 g
Sodium citrate, dihydratea 0.5-5 g
Magnesium sulfate, heptahydratea 1.0-10 g
FERMAX"" (antifoam) 0-5 ml
Ferric chloride, hexahydrate' 20-200 mg
Zinc sulfate, heptahydrate 0.2-20 mg
Cobalt chloride, hexahydrate' 0.2-20 mg
Sodium molybdate, dihydrate' 0.2-20 mg
Cupric sulfate, pentahydrate' 0.2-20 mg
Boric acid' 0.2-20 mg
Manganese sulfate, monohydratea 0.2-20 mg
Casein digest 15-25 g
Methionine' 0-5 g
Leucine' 0-5 g
-25-
CA 02528202 1999-10-21
' A portion of these ingredients was added to the fermentor initially, and the
remainder was fed during the fermentation. Ammonium hydroxide was added as
required to control pH.
The timing of arabinose addition ranged from 50 to 65 hours post-
inoculation. Bolus additions of 0.1% to it (final concentration) arabinose
were tested for the induction of co-expression of T4-lysozyme and endA
endonuclease.
The fermentation was allowed to proceed for about 65 hours (60 to 72
hours), after which the broth was harvested for subsequent treatment for
product recovery.
Assessment of Reduction of Broth Viscosity by endA Endonuclease Over-
expression: Aliquots of harvested broth from the rhuMAb CD18 F(ab')2-leucine
zipper fermentation with or without the co-expression of endA product in
addition to phage lysozyme described above was subjected to one cycle of
freeze-thaw. The thawed broth was diluted 1:3 into water or 20 mM MgC12 before
incubation in a 37 C water bath with agitation. Samples were removed at
intervals and the viscosity of the diluted broth was measured by using the
Falling Ball viscometer.
Results:
Effect of Over-Expression of EndA in Addition to T4-Lysozvme on Broth
Viscosity: As shown in Table 3, diluted freeze-thawed harvest broth from the
above fermentation process in the absence of endA over-expression had broth
viscosity in excess of 800 cP at the start of the 37 C incubation. After 60
minutes of incubation, there was little change in the H20-diluted freeze-
thawed
fermentation broth viscosity, while the MgCl2-buffer-diluted broth showed
significant reduction in the broth viscosity. Upon extended 37 C incubation
for up to over 2.5 hours, the viscosity of the freeze-thawed diluted harvest
broth with no over-expression of endA in addition to T4-lysozyme leveled off
at about 40 cP. The viscosity of the diluted freeze-thawed harvest broth with
over-expression of endA was less than 20 cP even before any 37 C incubation.
Table 3
Co-expression Treatment 37 C Incubation Broth Viscosity
(min) (cP)
T4-lysozyme + H2O control 0 <20
endA (pJJ154) + 20 mM MgCl2 0 <20
H2O control 60 <20
+ 20 mM MgC12 60 <20
T4-lysozyme only H2O control 0 >800
(pJJ153) + 20 mM MgC12 0 >800
H2O control 60 >800
+ 20 mM MgCl2 60 36
-26-
CA 02528202 1999-10-21
H2O control 120 41
+ 20 mM MgC12 120 36
H2O control 165 40
+ 20 mM MgC12 165 42
B. Co-expression of T4-Lvsozvme Gene t, and EndA Endonuclease:
Background:
As described in Example IA, over-expression of endA brings significant
benefit in lowering the viscosity of permeabilized or lysed broth. It helps
in conditioning the fermentation broth for the subsequent product recovery
step. By co-expressing T4-lysozyme and t-gene in addition to endA, cells can
be biochemically lysed and. at the same time yield a well-conditioned broth
lysate with fluid viscosity sufficiently low and compatible with product
isolation steps such as centrifugation or EBA.
The fermentation process described above was used to produce rhuMAb CD18
as a F(ab')2-leucine zipper precursor directed by plasmid pS1130 in E. coli,
with the co-expression of lytic enzymes and DNA-digesting protein directed by
the plasmid pJJ155. in E. coli. The antibody fragment product was secreted and
accumulated in the periplasm. The lytic enzymes were compartmentalized away
from their substrate until released by the action of the t-gene product. The
desired recovery process targets the soluble fraction of the F(ab')2-leucine
zipper released from the periplasm for initial capture.
Materials & Methods:
PJJ155 Plasmid Construction: Like pJJ154, pJJ155 is a pACYC177 derivative that
is compatible with pBR322 vectors. To construct pJJ155, pJJ154 as described
above was digested with KpnI and the vector fragment was ligated with PCR-
amplified t-gene designed to encode KpnI ends. The correct orientation of the
plasmid was screened for by restriction digest to product pJJ155. A map for
pJJ155 is shown in Fig. 7.
Bacterial Strains and Growth Conditions: Most experiments were carried out
with
transformed 33B8 as described above except that pJJ154 was replaced by pJJ155.
Fermentation Process Description: See Example IA.
Results:
Cell growth of 33B8 co-transformed with pS1130 and pJJ155
(33B8/pS1130/pJJ155) was not significantly different from that of the control
(33B8 transformed with pS1130 only). After addition of 0.5% to 1% arabinose
at 50-65 hours to induce the co-expression of the lytic enzymes and DNA-
digesting protein, OD550 of the 33B8/pSll30/pJJ155 culture steadily dropped to
30-4011 of peak cell density, suggesting cell lysis.
-27-
CA 02528202 1999-10-21
Table 4 shows the effect of co-expression of T4-lysozyme + endA + t-gene
on the release of soluble anti-CD18 antibody fragment into the medium. The
supernatant from centrifugation of the harvested broth lysate after incubation
in the presence of 25 mM EDTA was assayed by ion-exchange HPLC chromatography
for product quantitation. Greater than 80% of the soluble anti-CD18 antibody
fragments was found in the supernatant fraction for the experimental condition
where co-expression of lytic enzymes and DNA-digesting protein was induced,
compared to less than 10% found for the control and the condition with no t-
gene (pJJ154) or mechanical disruption.
Table 4
Co-Expression % of Total Product Released
None (control) <10
T4-Lysozyme + endA (pJJ154) <10
T4-Lysozyme + endA + t-gene (pJJ155) >80
Conclusions:
Endonuclease degrades DNA and lowers broth viscosity. Over-expression
of E. coli endogenous endonuclease in addition to T4-lysozyme reduces the need
for mechanical cell disruption for the shearing of released DNA. The release
of the phage lysozyme from the cytoplasmic compartment mediated by the
expressed t-gene protein initiates the biochemical disruption process,
resulting in cell lysis and the release of cellular contents including
heterologous polypeptide, genomic DNA, and the DNA-digesting protein, which
was
trapped in either the periplasm or the cytoplasm up to this time. By holding
the broth lysate for appropriate digestion of substrates by the lytic enzymes
and DNA-digesting protein co-expressed, the broth viscosity and product
release
from cellular matrix were improved for better product recovery.
EXAMPLE II
Co-expression of Lytic Enzymes with IGF-I
Background:
IGF-I was selected as a heterologous polypeptide for evaluation of
refractile particle recovery due to large-scale needs. Co-expression of lytic
enzymes and DNA-digesting protein was used to improve the release of the IGF-I
refractile particles from cell-wall structures in the absence of mechanical
disruption.
Upon cell lysis, in addition to releasing T4-lysozyme from the
cytoplasmic compartment, genomic DNA released from the cytoplasm would have
contributed significant viscosity to the broth lysate fluid. Hence, the
co-expression of an E.coli endonuclease together with lytic enzymes was useful
in reducing fluid viscosity following cell lysis and improving product
recovery
during centrifugation.
-28-
CA 02528202 1999-10-21
Materials & Methods:
pLBIGF57 Plasmid Construction: The plasmid pLBIGF57 for the expression of IGF-
I (Fig. 6) was constructed from a basic backbone of pBR322. The
transcriptional and translational sequences required for the expression of
nucleic acid encoding IGF-I were provided by the phoA promoter and tip Shine-
Dalgarno sequence. Secretion of the protein was directed by a TIR variant of
the lamB signal sequence. This TIR variant does not alter the primary amino
acid sequence of the lamB signal; however, silent nucleotide sequence changes
result, in this particular variant, in an increased level of translated
protein.
The details of pLBIGF57 construction follow. A codon library of the lama
signal sequence was constructed to screen for translational initiation region
(TIR) variants of differing strength. Specifically, the third position of
codons 3 to 7 of the lamB signal sequence was varied. This design conserved
the wild-type amino acid sequence and yet allowed for divergence within the
nucleotide sequence.
As previously described for the screening of the STII signal sequence
codon library (S. African Pat. No. ZA 96/1688; Simmons and Yansura, Nature
Biotechnology, 14: 629-634 (1996)), the phoA gene product served as a reporter
for the selection of the lamB TIR variants. The codon library of the lamB
signal sequence was inserted downstream of the phoA promoter and trp Shine-
Dalgarno and upstream of the phoA gene. Under conditions of low
transcriptional activity, the quantity of alkaline phosphatase secreted by
each
construct was now dependent on the efficiency of translational initiation
provided by each TIR variant in the library. Using this method, lamB TIR
variants were selected covering an approximate 10-fold activity range.
Specifically, lamB TIR variant #57 provides an approximately 1.8-fold stronger
TIR than the wild-type lamB codons based on the phoA activity assay.
The vector fragment for the construction of pLBIGF57 was generated by
digesting pBK131Ran with XbaI and Sphl. This XbaI-SphI vector contains the
phoA promoter and trp Shine-Dalgarno sequences. The coding sequences for IGF-I
and the lambda t, transcription terminator were isolated from pBKIGF-2B (U.S.
Pat. No. 5,342,763) following digestion with Ncol-SphI. The lamB signal
sequence fragment was isolated from pLBPhoTBK#57 (TIR variant #57; generated
as described above) following digestion with XbaI-NcoI. These three fragments
were then ligated together to construct pLBIGF57.
Bacterial Strains and Growth Conditions: Most experiments were carried out
with strain 43E7 (S. coli W3110 fhuA(tonA) d(argF-lac) ptr3 degP41(kanS)
GompTd(nmpc-fepR) ilvG+ phaA+). A double-plasmid system involving the product
plasmid (pLBIGF57) and pJJ155 for the lytic enzymes was employed. Competent
cells of 43E7 were co-transformed with pLBIGF57 and pJJ155 using the standard
procedure. Transformants were picked after growth on an LB plate containing
-29-
CA 02528202 1999-10-21
50 g/mL carbenicillin (LB + CARB50'") and 50 ug/ml kanamycin, streak-purified
and grown in LB broth with 50 pg/mL CARB50T and 50 ug/ml kanamycin in a 37 C
shaker/incubator before being tested in the fermentor.
For comparison, 43E7 transformed with pLBIGF57 alone was used in the
control case conducted under similar conditions. pLBIGF57 confers both
carbenicillin and tetracycline resistance to the production host and allows
43E7/pLBIGF57 to grow in the presence of either antibiotic.
Fermentation Process: The fermentation medium composition and experimental
protocol used for the co-expression of nucleic acid encoding IGF-I, endA,
T4-lysozyme, and t-gene if used were similar to those of the scaled-down
high-metabolic rate, high-yield 10-kiloliter IGF-I process. Briefly, a shake
flask seed culture of 43E7/pLBIGF57 or 43E7/pLBIGF57/pJJ155 was used to
inoculate the rich production medium. The composition of the medium (with the
quantities of each component utilized per liter of initial medium) is
described
below:
Ingredient Ouantity/L
Glucose* 200-500 g
Ammonium Sulfate 2-10 g ,
Sodium Phosphate, Monobasic Dihydrate 1-5 g
Potassium Phosphate, Dibasic 1-5 g
Sodium Citrate, Dihydrate 0.5-5 g
Potassium Chloride 0.5-5 g
Magnesium Sulfate, Heptahydrate 0.5-5 g
PLURONIC'"' Polyol, L61 0.1-5 mL
Ferric Chloride, Heptahydrate 10-100 mg
Zinc Sulfate, Heptahydrate 0.1-10 mg
Cobalt Chloride, Hexahydrate 0.1-10 mg
Sodium Molybdate, Dihydrate 0.1-10 mg
Cupric Sulfate, Pentahydrate 0.1-10 mg
Boric Acid 0.1-10 mg
Manganese Sulfate, Monohydrate 0.1-10 mg
Hydrochloric Acid 10-100 mg
Tetracycline 4-30 mg
Yeast Extract* 5-25 g
NZ Amine AS* 5-25 g
-30-
CA 02528202 1999-10-21
Methionine* 0-5 g
Ammonium Hydroxide as required to
control pH
Sulfuric Acid as required to
control pH
*A portion of the glucose, yeast extract, NZ Amine AS, and methionine is added
l0 to the medium initially, with the remainder being fed throughout the
fermentation.
The fermentation was a fed-batch process with fermentation parameters set
as follow:
Agitation: Initially at 800 RPM, increased to 1000
RPM at 8 OD
Aeration: 15.0 alpm
pH control: 7.3
Temp.: 37 C
Back pressure: 0.7 bar
Glucose feed: computer-controlled using an algorithm
which regulates the growth rate at
approximately 95% of the maximum early
in the fermentation and which then
controls the dissolved oxygen
concentration (DO2) at 30% of air
saturation after the DOz drops to 30t.
Complex nitrogen feed: constant feed rate of 0.5 mL/min
throughout the run
Run Duration: 40 hours
The timing of arabinose addition ranged from 24 hr to 36 hr. Bolus
additions of 0.1% to It (final concentration) arabinose were tested to define
the induction strength necessary for producing the most preferred amounts of
T4-lysozyme for better product recovery at the centrifugation step.
Recovery of Refractile Particles from Harvested Broth: Broth was harvested at
the end of fermentation when a target drop in OD550 was observed and was
either
processed soon after or stored briefly at 4 C prior to use. The test protocol
used involved four process steps:
I. Add IM EDTA to the harvest broth to bring the final concentration of
EDTA to 25 mM. EDTA chelates the divalent cations and disrupts the outer cell
surface structure. This makes the peptidoglycan layer inside unbroken cells
accessible to degradation by T4-lysozyme and weakens the cell wall to promote
cell lysis.
-31-
CA 02528202 1999-10-21
11. Hold the lysate at room temperature or incubate at 370C for further
degradation of cell wall. This step simulates the longer process times
associated with the larger-scale process.
111. Recover refractile particles and solids from the lysate by
centrifugation. Bench-scale centrifugation in a SORVALL" GSA rotor at
different speeds (3000 rpm to 6000 rpm; equivalent to RCF's of approximately
2500 g to 6000 g at rmax, respectively) was used to collect the solids as
pellets.
An additional step to wash the pellet with buffer would remove the lysate
entrained by the pellet and minimize the amount of contaminating E. coli
proteins in the refractile particle preparations.
Samples of the supernatant and pellet from centrifugation of broth lysate
resuspended in buffer were evaluated for product recovery. The amount of
product present in the samples was analyzed by a HPLC reverse-phase method.
Product recovery efficiency was calculated by expressing the amount of product
recovered in the pellet by the process step as a percent of the total product
present in the pellet and supernatant combined. To evaluate the quality of the
refractile particles recovered, the amount of total protein present in the
pellet and the supernatant was measured by the Lowry method (J. Biol. Chem.,
193: 265 (1951)).
The contribution of endonuclease activity was assessed by the efficiency
of solids recovery during sedimentation from the broth lysate by
centrifugation. Also, the amount of nucleic acids present in the pellet and
the supernatant was measured by OD260 readings.
Results:
IGF-I Fermentation and Product Expression:
Figure 8 shows in general the two-plasmid system employed in this Example
for co-expression of lytic enzymes and endA with IGF-I using pJJ155. The
initial growth rate of 43E7/pLBIGF57/pJJ155 showed no significant difference
from that of the 43E7/pLBIGF57 control. Peak cell densities reached in these
broths were similar. However, compared to the control, a significant loss in
optical density was observed in cultures after induction for lytic enzyme
co-expression, indicating cell lysis. Examination of the harvest broth by
phase-contrast microscopy showed that, in comparison to the no-co-expression
control, very few intact E. coli cells were present and freed refractile
particles were evident as a result of the co-expression of lytic enzymes and
DNA-digesting protein. See Figures 9A-9E.
The respiration rates across this collection of runs looked very similar
to the control except for significant continuous loss in oxygen uptake rate
(ouR) with a concomitant loss in kla soon after the arabinose addition.
The success of the biochemical cell lysis technique as described in this
invention is evident from the differences in the partitioning of nucleic acids
and total protein between the solid (pellet) and liquid (supernatant)
fractions
-32-
CA 02528202 1999-10-21
as a result of the co-expression of the lytic enzymes and DNA-digesting
protein
versus the control with no co-expression (Figs. 10A and 10B, respectively).
The percent of total nucleic acids calculated from A260 readings and the
percent of total protein as measured by the Lowry protein assay both increased
in the supernatant from the centrifugation of biochemically lysed broth over
that from control broth.
The product recovery from the two conditions is summarized in Fig. 11.
With the biochemically-lysed IGF-I broth, IGF-I product was released together
with degraded DNA polymer into the broth lysate. The efficiency of recovering
the small dense refractile particles increased with the RCF used during
centrifugation. As higher g force was used, the percent of the lysed broth
recovered as pellet (reported as % pellet) increased (Fig. 12), and so did the
amount of IGF-I product in the pellet. At approximately 6000 x g, close to 95%
of the product was captured in the pellet.
Conclusion:
As disclosed herein, a simple manipulation of gene expression during the
fermentation process resulted in a biochemical cell lysis that could replace
the conventional mechanical disruption traditionally used for product recovery
at production scale. In vivo co-expression or coordinated expression of T4-
lysozyme and t-gene product is a highly effective technique for the
disintegration of cells while the over-expressed endAprotein degrades the
leaked DNA, lowers broth viscosity, and efficiently conditions the broth
lysate
for product recovery in the initial product capture step. Biochemical cell
lysis is applicable to the recovery of soluble as well as insoluble product.
The compartmentalization of the co-expressed enzymes away from their
substrates
until the desired moment for cell lysis is essential and a critical design in
the invention. The invention brings significant reduction in process cost,
process time, and hence opportunity cost to other products that may be sharing
the same production facility.
-33-
CA 02528202 1999-10-21
DEirAA,NDES OU BREVETS VOLU-MINEUX
LA PRESENTE PARTIE DE CETTE DEIL-~NDE OLl CE BREVETS
COMPREND PLUS D'LIN TOME.
CECI EST LE TOME DE
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME OF
NOTE: For additional volumes please contact the Canadian Patent Office.
