Note: Descriptions are shown in the official language in which they were submitted.
0050/49041 CA 02329144 2000 - 11- 17
NEW PEPTIDE FRAGMENTS FOR PROTEIN PURIFICATION
The invention relates to novel peptide fragments, fusion proteins
comprising the peptide fragments, processes for preparing them,
and the use of the peptide fragments. The invention also relates
to a process for purifying fusion proteins and a method for
detecting proteins.
The invention further relates to nucleic acids which code for the
peptide fragments or for the fusion proteins, and vectors which
comprise the nucleic acids.
It has become possible through modern molecular biology to
prepare almost any proteins, peptides or their derivatives in
virtually unlimited quantities. In this connection, purification
of proteins frequently proves to be the limiting and not
uncommonly inefficient and thus eventually cost-determining
factor.
This is why a number of techniques for purifying proteins have
been developed. The techniques normally used to purify proteins
from cell supernatants, crude cell extracts or cells are, for
example, salt precipitation or precipitation with organic.
solvents, ultrafiltration, dialysis, gel electrophoresis,
isoelectric focusing, chromatofocusing, ion exchange or gel
chromatography, hydrophobic chromatography, immunoprecipitation
or IMAC (= immobilized metal affinity chromatography). Single
methods can be used for the purification, but a combination of
different techniques is usually necessary. Conventional
purification methods are reviewed, for example, in the textbooks
Protein Purification Process Engineering (Ed. R.G. Harrison,
1994, Marcel Dekker, Inc., New York, page 209 - 316, ISBN
0-8247-9009-X), Protein Purification (Ed. R.K. Scopes, 1994,
Springer Verlag New York, chapter 4 - 7, ISBN 0-387-94072-3) and
Methods for Protein Analysis (Ed. R.A. Copeland, 1994 Chapman &
Hall, page 59 - 112, ISBN 0-412-03741-6). It is important for
the purification of proteins that the purification takes place
under conditions which are as mild as possible, and is as
selective as possible and as quick as possible. Moreover the
protein losses should be kept as small as possible. Many of the
protein purification methods are insufficiently selective,
suitable for only small quantities of protein and/or very costly.
The method for purifying proteins by IMAC (= immobilized metal
affinity chromatography) described by Porath et al. (Nature,
Vol.258, 1975: 598 - 599) is a good compromise between the stated
requirements to be met by optimal purification. However, this
0050/49041 CA 02329144 2000-11-17
2
method still has some disadvantages. Thus, for example, not all
metal ions bind equally well to the support material so that some
of the ions are washed out and thus contaminate the required
product. Many proteins do not bind at all to the chromatography
material and thus cannot be purified, or bind too weakly so that
they are eluted even during the necessary steps for washing the
column material. This results in undesired losses of product.
Since the selectivity is usually inadequate for a one-step
purification, in contrast to purifications by biospecific
affinity purification methods such as, for example, purification
via antibodies, further purification steps are necessary.
In order to be able to purify a wider range of proteins using
this method, various so-called tags have been developed, such as
polyhistidine, His-Trp, His-Tyr or (His-Asp)n (see Sporeno et al.,
J. Biol. Chem. 269 (15), 1994: 10991 - 10995, Le Grice et al.,
Eur. J. Biochem., 187 (2), 1990: 307 - 314, Reece et al., Gene,
126 (1), 1993: 105 - 107, De Vos et al., Nucl. Acid. Res., 22
(7), 1994: 1161 - 1166, Feng et al., J. Biol. Chem. 269 (3),
1994: 2342 - 2348, Hochuli et al., Biotechnology, 1988: 1321 -
1325, Patwardhan et al., J. Chromatography A, 787, 1997: 91 -
100, Hutchens et al., J. Chromatography, 604, 1992: 133 - 141).
These tags are linked to the protein to be purified by means of
molecular biology at the nucleic acid level. It has been possible
through these tags to improve protein purification further in
some areas. However, even this method still has some disad-
vantages. It is still not possible to predict reliably whether a
protein can be purified by this method (see Immobilized Metal Ion
Affinity Chromatography, L. K&gedal, page 227 - 251 in Protein
Purification, Eds. J.C. Janson, L. Ryd6n, 1989, VCH Publishers,
Inc., New York, ISBN 0-89573-122-3), which means that this method
is not applicable to every protein either. Once again, metal ions
may be washed out or proteins may bind so weakly that they are
partly lost during the washing steps. The selectivity is still
unsatisfactory too. In addition, the capacity of the column
material for loading with the proteins to be purified is still in
some cases too low, so that a large amount of column material
must be used for a purification. The protein yield is also still
inadequate. This leads to unnecessary costs.
Volz et al. have described the purification of ATPases from
Helicobacter pylori without using an additional His-tag sequence.
These ATPases contain natural metal binding sites which make
purification by IMAC possible. Our own studies have shown that
these binding sites display a binding affinity which is too low
for efficient purification of all desired proteins.
0050/49041 New Iyage02329144 2000 11 17
. .
3
it is an object of the present invention to provide further tags
for protein purification by IMAC which do not have the
abovementioned disadvantages and thus make it possible, for
example, to use the tags more widely and/or load the column
material with a greater density, and which show a higher
selectivity and thus simplify the purification.
We have found that this object is achieved by the peptide
fragments according to the invention having the general sequence
H1S-X1-Hls-X2-X3-X4-CyS-X5-X6-CyS,
where the variables X1 to X6 in the sequence have the following
meanings:
X1 = an amino acid selected from the group of Ala, Val, Phe, Ser,
Met, Trp, Tyr, Asn, Asp or Lys and the variables X2 to X6 an
amino acid selected from the group of Gly, Ala, Val, Leu,
Ile, Phe, Pro, Ser, Thr, Cys, Met, Trp, Tyr, Asn, Gln, Asp,
Glu, Lys, Arg, His or
X2 = an amino acid selected from the group of Val, Ile, Phe, Pro,
Trp, Tyr, Gln, Glu or Arg and the variables X1, X3 to X6 an
amino acid selected from the group of Gly, Ala, Val, Leu,
Ile, Phe, Pro, Ser, Thr, Cys, Met, Trp, Tyr, Asn, Gln, Asp,
Glu, Lys, Arg, His or
X3 = an amino acid selected from the group of Gly, Ile, Thr, Met,
Trp, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His and the variables
X1, X2, X4 to X6 an amino acid selected from the group of Gly,
Ala, Val, Leu, Ile, Phe, Pro, Ser, Thr, Cys, Met, Trp, Tyr,
Asn, Gln, Asp, Glu, Lys, Arg, His or
X4 = an amino acid selected from the group of Val, Phe, Pro, Cys,
Met, Trp, Asn, Glu, Arg or His and the variables X1 to X3, X5,
X6 an amino acid selected from the group of Gly, Ala, Val,
Leu, Ile, Phe, Pro, Ser, Thr, Cys, Met, Trp, Tyr, Asn, Gln,
Asp, Glu, Lys, Arg, His or
X5 = an amino acid selected from the group of Gly, Ser, Cys, Met,
Trp, Asn, Glu, Lys or Arg and the variables X1 to X4, X6 an
amino acid selected from the group of Gly, Ala, Val, Leu,
Ile, Phe, Pro, Ser, Thr, Cys, Met, Trp, Tyr, Asn, Gin, Asp,
Glu, Lys, Arg, His or
CA 02329144 2000-11-17
0050/49041 New page
4
X6 = an amino acid selected from the group of Phe, Pro, Ser, Cys,
Trp, Tyr or Gln and the variables X1 to X5 an amino acid
selected from the group of Gly, Ala, Val, Leu, Ile, Phe, Pro,
Ser, Thr, Cys, Met, Trp, Tyr, Asn, Gin, Asp, Glu, Lys, Arg,
His and
where at least one of the variabes X1 to X6 in the sequence is,
independently of one another, Gln or Asn.
The general sequence His-X1-His-X2-X3-X4-Cys-X5-X6-Cys corresponds
to SEQ ID No: 1 where X1 corresponds to the amino acids designated
xaa in position 2 in SEQ ID NO: 1, and X2 corresponds to Xaa in
position 4, X3 corresponds to Xaa in position 5, X4 corresponds to
Xaa in position 6, X5 corresponds to Xaa in position 8 and X6
corresponds to Xaa in position 9. The amino acids mentioned above
for X1 to X6 may represent the corresponding amino acids
designated Xaa in SEQ ID NO: 1.
It is advantageous for at least one of the variables X1 to X6 in
the sequence additionally to be, independently of one another,
Lys or Arg. Further advantageous amino acids present in the
variables XI to X6 are Glu, Lys, Arg, Tyr, Cys, Lys, His, Asp or
Met. The amino acids Cys, Glu, Lys, Tyr or Arg are preferably
present, particularly preferably Cys. These amino acids
contribute to advantageous binding of the peptide fragments to
the immobilized metal ions. In addition, it is advantageous for
not more than four, preferably three, histidine residues to be
present consecutively in the sequence.
The variables X1 to X6 in the sequence have the further
advantageous and preferred meanings, independently of one
another:
X1 = an amino acid selected from the group of Ala, Val, Phe, Ser,
Met, Trp, Tyr, Asn, Asp or Lys, particularly preferably Phe,
Ser, Asn, Asp or Lys, very particularly preferably Asn;
X2 = an amino acid selected from the group of Val, Ile, Phe, Pro,
Trp, Tyr, Gln, Glu or Arg, particularly preferably Val, Ile,
Phe, Pro, Gln, Glu or Arg, very particularly preferably Gln,
Glu or Arg;
X3 = an amino acid selected from the group of Gly, Ile, Thr, Met,
Trp, Tyr, Asn, Gln, Asp, Glu, Lys, Arg or His, particularly
preferably Gly, Ile, Thr, Met, Trp, Tyr, Asn, Asp, Glu, Arg
or His, very particularly preferably Gly, Thr or Tyr;
CA 02329144 2008-06-17
X4 = an amino acid selected from the group of Val, Phe, Pro, Cys,
Met, Trp, Asn, Glu, Arg or His, particularly preferably Val,
Phe, Cys, Met, Trp, Asn, Arg or His, very particularly
preferably Asn or Arg;
X5 = an amino acid selected from the group of Gly, Ser, Cys, Met,
Trp, Asn, Glu, Lys or Arg, particularly preferably Gly, Ser,
Cys, Met, Asn, Glu, Lys or Arg, very particularly preferably
Gly or Lys;
X6 = an amino acid selected from the group of Phe, Pro, Ser, Cys,
Trp, Tyr or Gln, particularly preferably Phe, Ser, Cys or
Tyr, very particularly preferably Cys.
The variables X1 to X6 in the sequence His-X1-His-X2-X3-X4-Cys-XS-
X6-Cys may have the various preferred meanings independently of
one another, with individual variables up to a maximum of five of
the variables being an amino acid selected from the group of Gly,
Ala, Val, Leu, Ile, Phe, Pro, Ser, Thr, Cys, Met, Trp, Tyr, Asn,
Gln, Asp, Glu, Lys, Psg, His.
Therefore, the present invention concerns a peptide fragment comprising the
sequence:
His-Xl-His-X2-X3-X4-Cys-X5-X6-Cys,
where the variables Xl to X6 in the sequence have the following meanings
independently of one another:
X1 = Asn;
X2 = Gin, Glu or Arg;
X3 = Gly, Thr or Tyr;
X4 = Asn or Arg;
X5 = Gly or Lys; and
X6 = Cys.
Particularly preferred peptide fragments are fragments having the
sequences
His-Gln-His-Glu-Gly-Arg-Cys-Lys-Glu-Cys
His-Asn-His-Arg-Tyr-Gly-Cys-Gly-Cys-Cys
CA 02329144 2007-05-14
5a
His-Arg-His-Gly-Thr-Asn-Cys-Leu-Lys-Cys
His-Ile-His-Gln-Ser-Asn-Cys-Gln-Val-Cys.
The stated sequences correspond in each case to the sequences SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
The abovementioned protein fragment sequence is encoded by the
nucleic acid fragments according to the invention. Account must
be taken of the degenerate genetic code in this connection. The
nucleic acid fragments according to the invention may in
principle be present in any suitable nucleic acids. The nucleic
acid fragments are advantageously inserted into vectors in such a
way that it is possible to prepare composite nucleic acid
sequences (= gene constructs) which code for the fusion proteins
according to the invention. These gene constructs can, for
expression, advantageously be accommodated in a suitable host
organism which makes optimal expression of the fusion protein
possible. Suitable vectors are well known to the skilled worker
0050/49041 CA 02329144 2000-11-17
6
and can be found, for example in the book Cloning Vectors (Eds.
Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985,
ISBN 0 444 904018). Apart from plasmids, vectors mean all other
vectors known to the skilled worker, such as, for example,
phages, viruses, transposons, IS elements, plasmids, cosmids,
linear or circular DNA. These vectors may undergo autonomous
replication or chromosomal replication in the host organisms.
The nucleic acid sequences according to the invention mean
sequences which have been functionally linked to one or more
regulatory signals, advantageously to increase gene expression.
These sequences may be 3' and/or 5' terminal regulatory sequences
to enhance expression and are selected for optimal expression
depending on the selected host organism and gene. These
regulatory sequences are, for example, sequences to which
inducers or repressors bind and thus regulate the expression of
the nucleic acids. The gene construct may additionally
advantageously contain one or more so-called enhancer sequences
functionally linked to the promoter, and these make increased
expression of the nucleic acid sequence possible. This may take
place, for example, by an approved interaction between RNA
polymerase and DNA. It is also possible to insert additional
advantageous sequences at the 3' end of the DNA sequences, such
as other regulatory elements or terminators. The nucleic acid
fragments according to the invention are advantageously inserted
into the vector in such a way that they form the N-terminal
region of the fusion protein. However, they can also be located
at the C terminus, or else be located within the protein, but in
this case the function of the protein must not be affected, and
cutting out of the fusion protein is no longer possible.
The regulatory sequences are intended to make specific expression
of the genes and protein expression possible. This may mean, for
example, depending on the host organism that the gene is
expressed or overexpressed only after induction, or that it is
immediately expressed and/or overexpressed.
Advantageous regulatory sequences are present, for example, in
promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac,
lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, X-PR promoter or
in the X-PL promoter, which are advantageosly used in
Gram-negative bacteria. Further advantageous regulatory sequences
are, for example, present in the Gram-positive promoters amy and
SPO2, in the yeast or fungus promoters ADC1, MFa , AC, P-60,
CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV/35S,
SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or
phaseolin promoter. Also advantageous in this connection are the
promoters of pyruvate decarboxylase and of methanol oxidase froid,
0050/49041 CA 02329144 2000 - 11- 17
. .,
7
for example, Hansenula. It is also possible to use artificial
promoters for the regulation.
The regulatory sequences or factors may moreover preferably have
a beneficial effect on expression of the introduced gene, and
thus increase it. Thus, enhancement of the regulatory elements
can advantageously take place at the level of transcription by
using strong transcription signals such as said promoters and/or
enhancers. However, enhancement of translation is also possible
by, for example, improving the stability of the mRNA.
The nucleic acid sequences according to the invention
advantageously also contain signals which make it possible for
the proteins to be secreted into the medium or into cell
compartments. Examples of sequences of this type which may be
mentioned are the typical signal sequences such as, for example
the signal sequence of ompA (E. coli membrane protein). It is
additionally possible for other advantageous sequences to be
present, such as the seqeunce of the a factor, or for YACs
yeast artificial chromosomes) to be used.
The gene constructs (= nucleic acid sequences) according to the
invention advantageously additionally contain sequences which
make it possible to eliminate the protein fragments having the
abovementioned sequence according to the invention from the N or
C terminus of the fusion protein, preferably from the N terminus.
These sequences code, for example, for cleavage sites for a wide
variety of proteases such as, for example, for factor Xa,
enterokinase, human renin, carboxypeptidase A, thrombin, trypsin,
dipeptidyl peptidases, papain, plasmin, pepsin or other
proteases. Preferred cleavage sites are for factor Xa, human
renin, dipeptidyl peptidases, carboxypeptidase A or entero-
kinase, because these enzymes have a high specificity and thus
unwanted digestion of the protein to be purified can be avoided.
If other proteases are used, care must be taken that no cleavage
sites are inside the protein to be purified. The protein fragment
can also be deleted by cleavage with cyanogen bromide [e.g.
2-(2-nitrophenylsulfenyl)-3-bromo-3'-methylindolinium, hydroxyl-
amine etc.] in the presence of formic acid. However, in this case
refolding of the protein is usually necessary, which results in
this method being less preferred. Elimination of the protein
fragment by exoprotease digestion (under kinetic control) is also
possible. However, this usually results in product mixtures. The
cleavage sites preferably used are those making it possible to
delete the protein fragments without leaving residues of protein
fragments in the protein to be purified. if the protein fragments
according to the invention can be tolerated in the fusion proteip
0050/49041 CA 02329144 2000-11-17
8
without loss of function and without other disadvantages, a
specific site for detaching the protein fragment can be dispensed
with.
Suitable vectors are in principle all vectors which make
expression in pro- or eukaryotic cells possible. it is possible
in this connection to use vectors which replicate in only one
genus or those which replicate in several genera (called shuttle
vectors). Examples of advantageous vectors are plasmids such as
the E.coli plasmids pEGFP, pLG338, pACYC184, pBR322, pUC18,
pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290,
pIN-III113-B1, kgtll or pBdCI, preferably pEGFP, in Streptomyces
pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or
pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALSl, pIL2
or pBB116, in yeasts 2 m, pAG-1, YEp6, YEp13 or pEMBLYe23 or in
plants pLGV23, pGHlac+, pBIN19, pAC2004 or pDH51. Said plasmids
represent a small selection of the possible plasmids. Further
plasmids are well known to the skilled worker and can be found,
for example, in the abovementioned book Cloning Vectors (Eds.
Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985 ,
ISBN 0 444 904018).
In another embodiment of the vector, the nucleic acid sequence
according to the invention can also advantageously be introduced
in the form of a linear DNA into the microorganisms and be
integrated by heterologous or homologous recombination into the
genome of the host organism. This linear DNA can consist of a
linearized vector such as a plasmid or only of the nucleic acid,
i.e. the nucleic acid fragment and the gene for the protein
(= fusion protein gene), and, where appropriate, other regulatory
sequences.
Host organisms suitable for the gene construct according to the
invention are in principle all prokaryotic or eukaryotic
organisms. Host organisms which are advantageously used are
microorganisms such as Gram-positive or Gram-negative bacteria,
archaebacteria, fungi, yeasts, animal or plant cells such as
Drosophila, specifically D.melanogaster, mouse, zebra fish or
tobacco. Preferably used are Gram-positive or Gram-negative
bacteria, fungi or yeasts, particularly preferably the genera
Escherichia, Bacillus, Streptomyces, Aspergillus or
Saccharomyces, very particularly preferably E. coli.
Particular preference is given to the following combinations of
vector and host organisms such as Escherichia coli and its
plasmids and phages and the promoters belonging thereto, and
Bacillus and its plasmids and promoters, Streptomyces and its
plasmids and promoters, Aspergillus and its plasmids and
0050/49041 CA 02329144 2000 - 11- 17
~
. `,
9
promoters or Saccharomyces and its plasmids and promoters.
The fusion proteins according to the invention can be prepared as
described above in a process wherein the nucleic acid fragments
according to the invention, which code for the protein fragments
having the abovementioned sequence, are fused to a gene which
codes for the proteins to be purified and, where appropriate,
other advantageous sequences such as promoter and/or enhancer
sequences, cleavage sites for proteases etc. If necessary for
this purpose, a suitable restriction enzyme cleavage site is
introduced between the nucleic acid fragment and the gene of the
protein to be purified, and this construct is inserted via
suitable cleavage sites into a vector. Methods of this type are
known to the skilled worker and can be found for example, in the
textbooks by Sambrook, J. et al. (1989) Molecular cloning: A
laboratory manual, Cold Spring Harbor Laboratory Press, by F.M.
Ausubel et al. (1994) Current protocols in molecular biology,
John Wiley and Sons or D.M. Glover et al., DNA Cloning Vol.1,
(1995), IRL Press (ISBN 019-963476-9). Further advantageous
vectors are the Pichia pastoris vectors pPic and pGap. This yeast
is also a suitable host organism for the protein expression.
The protein fragments according to the invention are suitable for
preparing fusion proteins which can be purified easily, at low
cost and efficiently with the aid of the protein fragments. The
protein fragments and fusion proteins according to the invention
can thus be purified advantageously, very selectively and in good
yields and high purity. The protein fragments according to the
invention, and thus the fusion proteins prepared from them are
advantageously distinguished by binding to the immobilized metal
ions at least 1.5 times more strongly than the Helicobacter
pylori ATPase-439 sequence.
All proteins are suitable in principle for preparing the fusion
proteins. The proteins preferably used are those having a
biological effect in humans, animals or plants or those of
interest for organic synthesis. Examples thereof are proteins
such as enzymes, hormones, or storage or binding or transport
proteins. Examples which may be mentioned are proteins such as
hydrolases such as lipases, esterases, amidases, nitrilases,
proteases, mediators such as cytokines e.g. lymphokines such as
MIF, MAF, TNF, interleukins such as interleukin 1, interferons
such as y-interferon, tPA, hormones such as proteohormones,
glycohormones, oligo- or polypeptide hormones such as
vasopressin, endorphins, endostatin, angiostatin, growth factors,
erythropoietin, transcription factors, integrins such as GPIIb/
IIIa or avpIII, receptors such as the various glutamate receptors',
0050/49041 CA 02329144 2000 - 11- 17
*
angiogenesis factors such as angiotensin.
The process according to the invention for purifying fusion
proteins makes it possible, for example, to purify proteins from
5 natural sources such as plant or animal extracts, plant or animal
cell lysates, from culture media, fermentation broths or from
synthesis broths, to mention only a few by way of example.
The process according to the invention comprises the following
10 reaction steps:
a) bringing liquids which contain the fusion protein into
contact with immobilized metal ions in such a way that an
affinity linkage can form between the metal ions and the
fusion protein,
b) removing unbound substances present in the liquid,
c) eluting the bound fusion protein by abolishing the affinity
linkage by changing the liquid medium and
d) collecting the purified fusion protein.
The fusion protein is advantageously expressed in a suitable host
organism (see above) before the purification in order to increase
the yield of fusion protein. The host organism is cultured in a
suitable synthetic or complex medium which contains a carbon
source, a nitrogen source and, where appropriate, inorganic
salts, vitamins and trace elements, at a suitable temperature and
with suitable aeration.
Depending on whether the fusion protein is excreted from the
cells or not, the cells are first disrupted, and the cells or
cell detritus are advantageously removed. The methods used for
cell disruption are those known to the skilled worker, such as
ultrasound, French press, enzyme digestion, osmotic shock and
many others. The cells or cell detritus can be removed, for
example, by centrifugation or filtration. However, removal of the
cells or cell detritus is not absolutely necessary.
The liquid containing the fusion proteins is subsequently brought
into contact with the immobilized metal ions so that an affinity
linkage between the fusion protein and the metal ions can form.
The binding takes place at pH values greater than 7,
advantageously for example at pH 7.0 to 9.0, preferably between
pH 7.5 and 8Ø Advantageous buffers are single buffers or buffer
mixtures such as, for example, 50 to 1000 mM buffers such as
CA 02329144 2000-11-17
0050/49041
11
50 mM Tris/HC1 pH 8.0 + 150 mM NaCl, 100 mM sodium acetate pH 7.7
+ 500 mM NaCl, 20 mM sodium phosphate pH 7.7 + 500 mM NaC1 or
50 mM Tris/HC1 pH 8.0 + 150 mM NH4C1. These buffers make it
possible to load the fusion proteins onto the immobilized metal
ions. In the simplest and particularly advantageous case, the
fusion proteins are brought into contact with the immobilized
metal ions directly in the buffer used for the disruption or in
the incubation medium. It is advantageous for the liquids and the
immobilized metal ions to be brought into contact with one
another in a conventional chromatography column. This facilitates
the removal of unbound substances, for example proteins, by
washing the column with a suitable buffer. Suitable buffers are
buffers which do not impair the binding of the protein fragments
according to the invention or of the fusion proteins to the metal
ions, and are able to remove impurities. Buffers of this type
preferably have a pH above pH 7, advantageously a pH between pH
7.0 and 9.0, preferably between pH 7.5 and 8Ø It is also
possible to purify batch mixtures, in which the immobilized metal
ions are placed in a vessel and then the liquids are added or
vice versa, in this way. The buffers which have been mentioned
can be used for these batch mixtures. The mixtures are
advantageously centrifuged or filtered between the individual
washing steps.
Support materials suitable in principle for immobilizing the
metal ions are all conventional ones which can easily be
derivatized, show no or only slight nonspecific adsorption, show
good physical, mechanical and chemical stability, and have a high
external and internal surface area. Suitable materials can be
obtained commercially, for example, from Pharmacia LKB, Sweden
(SepharoseTM6B or SuperoseTM), Pierce, USA (immobilized
iminodiacetic acid I or II, immobilized tris(carboxymethyl)-
ethylenediamine), Sigma, USA (immobilized iminodiacetic
acid-agarose), Boehringer Mannheim, Germany (zinc chelate-
agarose) or Toyo Soda, Japan (TSKge1 Chelate-5PW). EP-B-0 253 303
describes further suitable materials. Further suitable and
advantageous materials are materials such as Ni-coated microtiter
plates (nickel-chelate coated Flashplate , NEN life science pro-
ducts) or magnetic particles or specifically metal ion-treated
and binding membranes.
The various metal ions are bound in suitable materials
advantageously via groups such as IDA (= iminodiacetic acid), NTA
(= nitrilotriacetic acid) or TED (= tris(carboxymethyl)-
ethylenediamine). Suitable metal ions are Co, Cu, Fe, Ca, Mg, Ni,
Al, Cd, Hg or Zn, preferably Fe, Ni or Cu, particularly
preferably Ni or Cu, very particularly preferably Ni. The loadin!g
CA 02329144 2007-05-14
12
of the materials with metal ions advantageously takes place with
0.1 to 0.4 M~olutions of the metal salts in aqueous, unbuffered
solution.
After the washing, the fusion protein is eluted with a suitable
buffer. This buffer abolishes the affinity linkage between the
fusion protein and the immobilized metal ions. The fusion
proteins can be eluted via a pH gradient (low pH values < pH 7.0
act to elute), competitive ligands such as imidazole, organic
solvents such as acetone or ethanol, chelating agents such as
EDTA or NTA and/or detergents such as Tween*80. Elution via
competitive ligands such as imidazole and/or detergents is
preferred. Imidazole is used for the elution in a range from 0.05
to 0.7 IM, preferably from 0.1 to 0.5 M. The competitive ligands
and/or detergents are advantageously employed in a buffer, but
can also be used in water. Advantageous buffers are buffers which
correspond to the buffers used to load onto the immobilized metal
ions. This has the advantage that no unwanted interactions
between the column material, the bound proteins and the buffer
occur. Advantageous buffers preferably have a pH greater than
pH 7, advantageously a pH between pH 7.0 and 9.0, preferably
between pH 7.5 and B.O. These buffers are preferably applied via
an increasing gradient. In the case where a pH gradient is used
for the elution it is possible to use buffers with a pH below pH
7.0 and/or acids. The eluted fusion protein is collected and can
be used immediately or else, if necessary and if desired, treated
further. Suitable loading and elution buffers are to be found,
for example, in the textbook Protein Purification (Eds. J.C.
Janson, L. Ryden, VCH Publisher Inc., 1989, pages 227 to 251).
The protein fragment according to the invention can be deleted
using the methods described above, such as cyanogen bromide or
protease cleavage. It is possible in this case for residues of
the protein fragment to remain in the molecule or else to be
completely eliminated from the protein to be purified. The
protein fragment is advantageously removed completely from the
protein.
Suitable and advantageous protein fragments for preparing fusion proteins can
be screened by the following process according to the invention. As such, the
invention also concerns a process for preparing protein fragments able to
enter
* trademark
CA 02329144 2007-05-14
13
into a reversible affinity linkage with immobilized metal ions, which-
comprises
carrying out the following steps:
a) preparing a nucleic acid library starting from any suitable nucleic acid
sequence which codes for a peptide fragment consisting of the sequence:
His-Xl-His-X2-X3-X4-Cys-X5-X6-Cys,
where the histidine and cysteine residues of the sequence are conserved in the
nucleic acid library, and where the variables X1 to X6 in the sequence have
the
following meanings independently of one another:
Xl = Asn;
X2 = Gin, Glu or Arg;
X3 = Gly, Thr or Tyr;
X4 = Asn or Arg;
X5 = Gly or Lys; and
X6 = Cys;
b) fusing the nucleic acids of the library to a reporter gene which makes it
possible to detect the fusion protein encoded by the resulting nucleic acid
via its
binding to the immobilized metal ions and
c) selecting the nucleic acid sequences which display a reversible binding to
the immobilized metal ions which is at least 1.5 times stronger than the
binding
of the protein fragment encoded by the nucleic acid sequence in the natural
Helicobacter pylori ATPase-439.
The nucleic acid library based on the abovementioned sequence can
be constructed by methods for mutagenesis known to the skilled
worker. For this purpose, the sequence can be subjected, for
example, to a site directed mutagenesis as described in D.M.
Glover et al., DNA Cloning Vol.1, (1995), IRL Press
(ISBN 019-963476-9), Chapter 6, pages 193 et seq.
Spee et al. (Nucleic Acids Research, Vol. 21, No. 3, 1993: 777 -
778) describe a PCR method using dITP for random mutagenesis.
CA 02329144 2007-05-14
13a
The use of an in vitro recombination technique for molecular
evolution is described by Stemmer (Proc. Natl. Acad. Sci. USA,
Vol. 91, 1994: 10747 - 10751).
Moore et al. (Nature Biotechnology Vol. 14, 1996: 458 - 467)
describe combination of the PCR method and recombination method.
The use of an in vitro recombination technique for molecular
evolution is described by Stemmer (Proc. Natl. Acad. Sci. USA,
Vol. 91, 1994: 10747 - 10751). It is also possible to use a
combination of the two methods.
The use of mutated strains with defects in the DNA repair system
is described by Bornscheuer et al. (Strategies, 11, 1998: 16 -
17). Rellos et al. describe a PCR method using non-equimolar
amounts of nucleotides (Protein Expression and Purification, 5,
1994: 270 - 277).
0050/49041 CA 02329144 2000 - 11- 17
14
The nucleic acid library can advantageously be produced by a PCR
technique using two complementary, degenerate oligonucleotides
(called wobble primers), as described in the examples. It is
important that the histidine and cysteine residues present in
this sequence are conserved.
For the screening for protein fragments having an improved
metal-binding affinity, the nucleic acid fragment according to
the invention, which codes for the protein fragment, is fused to
a reporter gene. Advantageous reporter genes make it easy to
detect the binding to the immobilized metal ions via, for
example, binding of antibodies which are labeled with a
fluorescent dye and are directed against the reporter gene, or
via self-fluorescing proteins such as the advantageous and
preferred egf protein from E.coli (= green fluorescent protein,
see Prasher et al., Gene 111 (2), 1992: 229 - 233) or the
preferred bioluminescence protein from Aequoria victoria or via
light-generating proteins such as the luciferin/luciferase
system. Particularly preferably used is a gfp protein mutant
(= egfp = enhanced green fluorescence protein) with a 35-fold
higher fluorescence activity caused by two point mutations at
position 64, replacement of Phe by Leu, and position 65,
replacement of Ser by Thr. This protein mutant has the advantage
over the wild-type protein that it is soluble and forms no
inclusion bodies. The use of the egfp protein makes it possible
to locate and quantify the protein concentration in each phase of
the purification of the proteins without interfering with the
purification and without using other cofactors or substrates
(Poppenborg et al., J. Biotechnol., 58 (2), 1997, 77 - 88). The
egfp protein is also distinguished by a high stability toward a
wide pH range (pH 5.5 to 12), bleaching by photooxidation,
oxidizing and weakly reducing agents such as 2% mercaptoethanol.
The protein shows a decrease in fluorescence above 37 C. Likewise
suitable as reporter gene are the gfp-uv (blue fluorescence) and
the eyfp (yellow fluorescence) proteins.
The suitable sequences are selected by comparing with the binding
affinity to the immobilized metal ions of the following natural
Helicobacter pylori ATPase-439 sequence His-Ile-His-Asn-Leu-Asp-
Cys-Pro-Asp-Cys. The protein fragment sequences according to the
invention show a reversible binding to the immobilized metal ions
which is at least 1.5 times stronger, preferably at least twice,
and particularly preferably at least three times, stronger.
Advantageous sequences make it possible for the protein yield
after the purification to be at least 20%, preferably at least
30%, particularly preferably at least 40%, very particularly
preferably at least 50%.
0050/49041 CA 02329144 2000 - 11- 17
The process according to the invention for screening the nucleic
acid library is advantageously suitable for automation. This
process can be used easily for testing a large number of nucleic
acid fragments and protein fragments for their metal ion binding
5 affinity in so-called high-throughput screening.
Proteins can easily be detected using the protein fragments
according to the invention. In the method according to the
invention for detecting proteins, individual proteins which
10 comprise a protein fragment having the abovementioned protein
fragments according to the invention in a protein mixture are
detected via antibodies which are directed against the protein
framework. Detection of these fusion proteins advantageously
takes place via mono- or polyclonal antibodies directed against
15 the protein fragment (= tag). The protein mixture can
advantageously be fractionated by chromatography or
electrophoresis before the detection and subsequently be
transferred (= blotted) to a suitable membrane (e.g. PVDF or
nitrocellulose) by conventional methods (see Sambrook et al.).
This membrane is then incubated with an antibody directed against
the tag. it is advantageous to wash the membrane several times
and then to detect the bound antibodies via a specific reaction
with a second antibody which is, for example, enzyme-conjugated
(e.g. alkaline phosphatase, peroxidase etc.) and is directed
against the constant region of the first, in a Western blot or
immuno blot. Corresponding antibodies are commercially available.
Where magnetic particles are used, the washing can be omitted and
the antibody-coated magnetic particles can be purified by fishing
out with magnets.
The protein fragments according to the invention have the
advantage over the conventional His tags for protein detection
that they have a stronger antigenic effect and thus are more
suitable for producing antibodies against the tag.
The invention is illustrated further by the following examples.
Examples:
The Chelating Sepharose Fast-Flow from Pharmacia LKB, Uppsala,
Schweden, was used for the test of binding to metal chelate
columns (= immobilized metal ions). Ampicillin, imidazole, EDTA
and all other reagents were purchased from Fluka (Buchs,
Switzerland). The DNA gel extraction kit, the Midi Plasmid-Kit
and the Prepspin Plasmid Kit originated from Qiagen (Hilden,
Germany), the restriction enzymes, the DNA-modifying enzymes, the
T4 DNA ligase and the Taq polymerase originated from MBI
0050/49041 CA 02329144 2000 - 11- 17
16
Fermentas (St. Leon-Rot, Germany). The Taq Dye Cycle Sequencing
Kit was purchased from Applied Biosystems (Weiterstadt, Germany).
The E. coli strain DH5a (F- endAl hsdRl7 [rk-, mk+] supE44 thil
5%gyrA96 relAl 0(argF laczya) U169) was used for the cloning
experiments. The plasmids which contained the gene for the egf
protein were purchased from Clonetech USA. The E. coli strains
were cultured in Luria-Bertani medium (= LB) with 100 g/ml Ampi-
cillin at 37 C to select the clones for transformation with the
egfp vector. The lysis buffer contained 50 mM sodium phosphate
buffer pH 8.0, 300 mM NaCl, 1 mg/ml Lysozym and 1 mM PMSF
(= phenylmethanesulfonyl fluoride = specific trypsin and
chymotrypsin inhibitor).
The DNA methods, such as ligations, restrictions, PCR or
transformations etc., were carried out as described in Sambrook,
J. et al. (1989) Molecular cloning: A laboratory manual, Cold
Spring Harbor Laboratory Press or F.M. Ausubel et al. (1994) Cur-
rent protocols in molecular biology, John Wiley and Sons. The
fluorescence-labeled dideoxy-DNA sequencing method was used for
the sequencing. The DNA sequencing was carried out using the Taq
Dye DeoxyTM Cycle Sequencing Kit (Applied Biosystems) and the 373A
DNA Sequencing System (Applied Biosystems) in accordance with the
manufacturer's instructions.
Example 1: Preparation of randomly mutagenized N-terminal
metal-binding sites which were bound to the egfp
gene, and of his6-egfp
For the PCR, the plasmid egfp and the two following complementary
oligonucleotides
5'-GCA.ATACCATGGGGCATNNNCATNNNNNNNNNTGTNNNNNNTGTGTGAGGAAGGGCGAG-3'
5'-CAGTTGGAATTCTAGAG-3'
were used. In the case of his6-egfp, the following two
complementary primers
5'-GCAATACCATGGGGCATCATCATCATCATCATGTGAGGAAGGGCGAG-3'
5'-CAGTTGGAATTCTAGAG-3'
were used.
The conditions used for the PCR were as follows:
CA 02329144 2007-05-14
17
Mixture: 8 ul of dNTP mix (200 mol)
ul of 10 x ThermoPol* buffer (New England Biolabs)
1 ul (100 pmol) of primer 1
1 ul (100 pmol) of primer 2
1 l (100 ng) of egfp plasmid
79.5 ul of water
1 ul of Deep Vent* polymerase (New England Biolabs)
PCR program 95 C 7min
95 C lmin
56 C lmin 30x
72 C 3min
72 C 7min
The PCR products were each digested with NcoI and NotI and
ligated into the egfp vector which had been digested with the
same enzymes, in order to preclude mutations in the vector (see
Figure 1). The PCR-vector ligations were used to transform
E. coli. The transformants were plated out on LB agar with
100 ug/ml Ampicillin and incubated at 37 C.
Example 2: Cultivation conditions and preparation of the cell
lysates
Transformed colonies which showed fluorescence, and some which
showed no fluorescence, were selected and cultured in 50 ml of LB
medium which contained 100 ug/ml Ampicillin. Colonies showing
fluorescence were selected for the high-throughput screening and
were incubated in sterile microtiter plates which contained
250 ul of LB medium with 100 ug/ml Ampicillin. After incubation,
the cultures were centrifuged. The pellets were resuspended in
2 ml of lysis buffer, incubated on ice for 20 minutes and then
disrupted with ultrasound (twice, 5 minutes with a Branson Soni-
fier 250). After centrifugation (15 min, 4 C, 20000 x g) the
various egfp mutants were obtained in the supernatant. All the
selected clones were sequenced (see Tables I and II). Clones
which showed no fluorescence contained stop codons in the
sequence, so that no functional proteins were expressed (see
Table I, A8, A13, M16a, Z4, Z11 and Z13). To quantify the bound
proteins, a fluorescence measurement was carried out in all the
experiments and correlated in a wide range with the gfp
concentration (see Figure 2).
Example 3: Low-throughput screening with Ni-NTA columns from
Qiagen
' trademarks
0050/49041 CA 02329144 2000 - 11- 17
18
600 l of the lysed cells were loaded onto an Ni-NTA column,
washed twice with 600 l of a washing solution (50 mM sodium
phosphate buffer, pH 8.0, 250 mM NaCl) and then eluted with a
0.7 M imidazole solution.
Example 4: High-throughput screening with membrane filter plates
Membrane filter microtiter plates (MultiScreen 5 m; supplied by
Millipore, Molsheim, Germany) were used for the high-throughput
screening. 250 l of a stirred chelate Sepharose suspension were
placed in each well of the membrane filter plates three times.
After each, addition, the Sepharose was centrifuged down (2 min,
23 C, 350 rpm). All further steps were carried out in a Beckmann
Biomek 2000 robot. The minicolumns in the wells were washed twice
with 250 l of water. The Sepharose was then loaded with 250 l of
a metal salt solution and equilibrated three times with 200 l of
buffer (50 mM sodium phosphate, pH 8.0, 250 mM NaCl). 0.3 M NiC12,
0.3 M CuSO4 or 0.3 M ZnC12 solutions were used for each of the
various mixtures (loading with metal ions). Aqueous unbuffered
metal salt solutions were used. 250 l of the cell lysate
supernatants were placed on each of these minicolumns. The
columns were then washed twice with 250 l of equilibration
buffer. The bound proteins were eluted with 2 x 100 l of 0.5 M
imidazole in equilibration buffer. The chelate Sepharose in the
filters can be regenerated with 250 l of 50 mM EDTA, 1 M NaCl in
water and be used for further screening experiments.
Example 5: IMAC experiments
A conventional chromatography system consisting of a glass
column, two peristaltic pumps for applying the solutions, a UV
detector (LKB UV-MII), a printer (LKB RIC 102) and a fraction
collector (LKB FRAC-200) was chosen for the experiment. All the
apparatus originated from Pharmacia. The column was packed with
chelating Sepharose Fast-Flow Gel (Pharmacia), washed with 7 bed
volumes of deionized water and loaded with the metal ions with 7
bed volumes of 0.3 M NiC12 solution. The column was then washed
and equilibrated with 7 bed volumes of IMAC buffer (50 mM sodium
phosphate, pH 8.0, 250 mM NaCl). 1 ml samples of the cell lysates
were loaded onto the column at a flow rate of 1.5 ml/min and
washed with 10 bed volumes of IMAC buffer. The bound proteins
were eluted via an increasing gradient with 0.5% of a 0.5 M
imidazole solution per ml of elution solution and finally 5 bed
volumes of a 0.5 M imidazole/water solution. The protein
fractions were identified by UV detection and collected. After
completion, the column was washed and regenerated with 50 mM
EDTA/1 M NaCl solution. This washing step detaches the metal,
bound cell residues and proteins from the column. The eluted
0050/49041 CA 02329144 2000 - 11- 17
19
fractions were examined both optically and spectroscopically.
Some of the investigated clones showed no affinity for the matrix
(see Tab. I, M15, M16), while others showed good binding (see
Tab. I, M13, Z5 and Z7). The clones M13 and Z5 eluted from the
column in a sharp band, whereas there was smearing of clone Z7 on
the column.
Example 6: Experiment comparing egfp wild-type and his-tag
The clone M13 was compared with the egfp wild-type protein and
the usual his tags in a comparative experiment. The egfp
wild-type protein does not bind to the metal chelate columns.
Fluorescence was no longer detectable on the column after washing
the column. The clone M13 binds to the column in a sharp band,
whereas the his tag proteins bind over the entire column. This is
attributable to a lower affinity, which leads eventually to a
lower capacity of the column. The protein yield in the case of
M13 is 56%, which is higher than the 48% with the his tags.
Table I: Binding experiments with Ni metal chelate columns
Clone Amino acid sequence
A6 His Gln His Glu Gly Arg Cys Lys Glu Cys gfp
A8 His Cys His Pro Glu Leu Cys Stop Leu Cys gfp
A13 His Leu His Ser Ile Gly Cys Pro Stop Cys gfp
M13 His Asn His Arg Tyr Gly Cys Gly Cys Cys gfp
M14 His Ser His Ser Val Gly Cys Phe Phe Cys gfp
M15 His Gly His Thr Leu Ser Cys Gly Leu Cys gfp
M16 His Ser His Thr Leu Arg Cys Lys Gly Cys gfp
M16a His Ser His Stop Leu Arg Cys Lys Gly Cys gfp
Z4 His Stop His Asn Stop Val Cys Ala Thr Cys gfp
Z5 His Arg His Gly Thr Asn Cys Leu Lys Cys gfp
Z7 His Ile His Gln Ser Asn Cys Gln Val Cys gfp
Z11 His Thr His Ala Ser Gly Cys Stop Stop Cys gfp
Z13 His Cys His Thr Trp Cys Cys Asn Stop Cys gfp
The clones A6, A10, M13, Z5 and Z7 bind well to the metal chelate
column, whereas the clones M14, M15 and M16 showed no binding.
Table II: Further sequenced clones detected by fluorescence in
the High-throughput screening
CA 02329144 2000-11-17
0050/49041
Clone Amino acid sequence
Al His Gly His Met Glu Arg Cys Leu Val Cys gfp
A2 His Lys His Ala Arg Ser Cys Met Gly Cys gfp
5 A3 His Phe His Thr Val Phe Cys Phe Ser Cys gfp
A4 His Arg His Arg Gly Met Cys Thr Ala Cys gfp
A12 His Asp His Arg Gly Val Cys Gly Leu Cys gfp
A14 His Asp His Glu Arg Leu Cys His Asn Cys gfp
10 X8 His Gly His Gly Asn Arg Cys Cys Gly Cys gfp
X9 His Arg His Gly Thr Ala Cys Met Asp Cys gfp
X11 His Ile His Ile Met Thr Cys Leu Ser Cys gfp
X12 His Thr His Pro Arg Ser Cys Ala Glu Cys gfp
X15 His Gly His Asp Arg Thr Cys Arg Gly Cys gfp
15 X16 His Arg His Ala Ile Ser Cys Ile Gly Cys gfp
X17 His Ile His Arg Gly Asp Cys Tyr Glu Cys gfp
X18 His His His Gly Ser Thr Cys Pro Thr Cys gfp
X19 His His His Phe His Ser Cys Phe Tyr Cys gfp
20 Z8 His Lys His Val Asp His Cys Gly Arg Cys gfp
Z9 His Ser His Leu Thr Leu Cys Leu Gly Cys gfp
Z10 His Thr His Gln Ser Gln Cys Gly Arg Cys gfp
Z14 His Arg His Leu Phe Trp Cys Ser Glu Cys gfp
Example 7: Metal binding affinity
Many of the clones showed a preferred binding to Ni2+ or Cu2+. In
the case of M13, no binding to Zn2+ was observed. On use of Ni
chelate columns, the clone M13 showed distinctly better
purification of the proteins by comparison with the his tags.
Conversely, the latter resulted in a purer product by comparison
with M13 on use of Cu chelate columns, but, since the binding to
the column material is very strong in both cases, Cu ions were
washed out by the drastic elution conditions. This leads to
contamination of the products.
Example 8: Experiment comparing between the ATPase-439 sequence
and protein fragments according to the invention
The ATPase-439 comparison clone was carried out in analogy to
Example 1 and 6. The primer used was the following primer
5'-GCAATACCATGGGGCATATTCATAATCTTGATTGTCCTGATTGT-3'. The other
primers and the PCR conditions were as described in Example 1.
0050/49041 CA 02329144 2000 - 11- 17
;.
21
The experiment was carried out with a Qiagen Ni-NTA Spin Kit
under native conditions, lysis being carried out as described
under these conditions; the columns which had already been loaded
were equilibrated with 600 l of 50 mM sodium phosphate buffer,
pH 8.0, 300 mM NaCl and centrifuged at 2000 rpm (= 420 x g) for
2 min. Then 600 Rl of the lysate were applied and centrifuged for
2 min. Two washes were carried out with 600 l of a 50 mM sodium
phosphate buffer, pH 8.0, 300 mM NaCl each time. Then 600 l of
50 mM sodium phosphate buffer, pH 8.0, 300 mM NaCl, 0.5 M
imidazole were used for two elutions. The yield of pure protein
was 1.5 times higher with the clone M13 than the metal-binding
site of the ATPase-439 comparison clone. Clone M13 thus binds
better and can also be eluted better.
20
30
40
CA 02329144 2001-05-09
2329144.seq
SEQUENCE LISTING
<110> BASF Aktiengesellschaft
<120> New peptide fragments for protein purification
<130> 10090-2303
<140> 2.329.144
<141> 1999-05-20
<140> PCT/EP99/03469
<141> 1999-05-20
<150> DE 198 22 823.6
<151> 1998-05-20
<160> 5
<170> PatentIn Vers. 2.0
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<400> 1
His Xaa His Xaa Xaa Xaa Cys Xaa Xaa Cys
1 5 10
<210> 2
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<400> 2
His Gln His Glu Gly Arg Cys Lys Glu Cys
1 5 10
<210> 3
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<400> 3
His Asn His Arg Tyr Gly Cys Gly Cys Cys
1 5 10
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<400> 4
His Arg His Gly Thr Asn Cys Leu Lys Cys
1 5 10
<210> 5
<211> 10
<212> PRT
<213> Artificial Sequence
Page 1
CA 02329144 2001-05-09
2329144.seq
<220>
<400> 5
His Ile His Gln Ser Asn Cys Gln Val Cys
1 5 10
Page 2
