Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Obesity Protein Intermediates and Their Preparation and Use
The present invention is in the field of human
medicine, particularly in the treatment of obesity and
disorders associated with obesity. Most specifically the
invention relates to an intermediate used to prepare obesity
proteins that when administered to a patient regulate ~at
tissue.
The production of proteins in heterologous host
organisms leads to the formation o~ inactive, sparingly-
soluble protein aggregates, i.e., inclusion ~odies. The
~ormation of such lnclusion bodies is a result o~ the high
protein concentrations in the cell arising ~rom expression.
In order to obtain ~iologically-active proteins, the
inclusion bodies must be dissolved by denaturation and
reduced, and then the three-~;~n~ional structure of the
protein in its native spatial ~orm must be ~ormed by the
adjustment o~ suitable conditions. R. Jaenicke, Proa.
Rio~hYs. Molec. Biol. 49:117-237 (1987).
However, renaturation into the biologically active
confor~ation is not a quantitative process -- a multitude of
non-functional species and con~ormations of the protein may
be formed. In order to shi~t the equilibrium to form the
biologically active con~ormation, conditions are selected
which, both prevent the establishment of improper protein
conformation and do not hinder renaturation. Because it is
difficult, i~ not impossible, to predict how a polypeptide
chain renatures into a highly, ordered confor~ation, the
conditions that ~avor the formation of the biologically
active protein cannot ~e predicted.
Recently, Yiying Zhang and co-workers pl1hl;.~h~A
the positional cloning of the mouse gene linked with obesity
and aiabetes. Yiying Zhang et al. Nature 372: 425-32
(1994). ~his report disclosed a gene coding ~or a 167 amino
acia protein with a putative 21 amino acid signal peptide
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that is exclusively expressed in adipose tissue. This
peptide is speculated to be an adiposity regulating hormone.
Likewise, Murakami et al., in Bioc~e~. and Bio~hys. Res.
CQ~m. 209(3): 944-52 (1995) disclose the obese rat gene and
protein.
The present invention provides the penultimate
intermediate in the renaturation pathway for the obesity
protein. The invention further provides the preparation of
the intermediate and its use to prepare a biologically
active obesity protein or analog thereof. Most
significantly, by passing through this int~rme~;ate, a
substantial increase in yield o~ the biologically active
protein is observed. The int~rm~ te also provides
significant process advantages that allow the large scale
manufacture of an obesity protein.
The present invention provides a properly folded
intermediate of an obesity protein or analog thereof. More
particularly, the present invention is directed to a
properly folded protein o~ the Formula (I):
96 1~l
A H - CH -
H2 C~
Rl B
R2
S
H2 C~
HOOC C- NH
146 (I)
wherein:
A is a polypeptide consisting essentially o~ a~ino acid
residues 1 to 95 of an obesity protein or analog thereof;
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B is a polypeptide consisting essentially of amino acid
residues 97 to 145 of the protein;
Rl and R2 are independently H or in conjunction with
the sulfur to which it is bound ~orms a mixed disulfide;
provided that both Rl and R2 are not H.
The invention also provides a process o~ preparing
a protein of Formula I, which comprises: mixing an obesity
protein or analog thereof with a solution comprising a
denaturant and a thiol r~ c; ng reagent at a concentration
of about 1 to 100 mM at a pH ~rom about 7 to about 12.
,
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~ he invention further provides a process o~
preparing a properly folded protein o~ the Formula (II):
96
A N - CH C
H2 C~
HOOC- C- NH
146 (II)
wherein:
A is a polypeptide consisting essentially of amino acid
residues 1 to 95 o~ an obesity protein or analog thereo~;
B is a polypepti~e consisting essentially of amino acid
residues 97 to 145 o~ the protein;
which comprises:
(a) Solubilizing obesity protein inclusion bodies
in a solution comprising:
a denaturant at a concentration sufficient to
solubLlize the protein, and
a thiol reaucing reagent at a concentration o~
about 1 to 100 mM, at a pH from about 7 to about 12;
(b) Reducing the thiol and denaturant
concentration of the solution to e~ect disul~ide bond
~ormation.
~escription of the Fiqures
Figure 1 provides an HPLC chromatogra~ run on a Zorbax C-8
column with buffer A being 50 mM ~m~nn;um phosphate, O.5%
SDS, and 5 96 n-propanol at pH 7.6 ana bu~er B being 50 mM
ammoniu~ phosphate, 0.5% SDS, ana 50 ~ n-propanol at pH 7.6.
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The peak at about 385 seconds represents the biologically
active obesity protein and the later eluting peak at about
573 seconds represent the c-;~;m~ intermediate prepared in
the examples.
For purposes of the present invention, as
disclosed and claimed herein, the following terms and
abbreviations are defined as follows:
sase pair (bp) -- refers to DNA or RNA. The
abbreviations A,C,G, and T correspond to the 5'-
~onophosphate ~orms of the nucleotides (deoxy)adenine,
(deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine,
respectively, when they occur in DNA molecules. The
abbreviations U,C,G, ana T correspond to the 5~-
monophosphate ~orms of the nucleosides uracil, cytidine,
gll~n;ne, and thymine, respectively when they occur in RNA
molecules. In double stranded DNA, base pair may re~er to a
partnership of A with T or C with G. In a DNA/RNA
heteroduplex, base pair may refer to a partnership o~ T with
U or C with G.
Denaturing reagent or denaturant -- is known to
one skilled in the art. Examples of denaturing reagents are
described in R. Jaenicke, Pro~. ~io~hys. Molec. Biol. 4~:
117-237 (1987). Preferred reagents include urea,
thiocyanate, and guanidine, most preferably, 6 to 8 M urea.
Mixed disul~ide -- refers to a group derived from
a thiol reducing reagent capable of disul~ide ex~hAn~e
between the reagent and the cysteinyl residue o~ the
polypeptide.
Obesity protein -- refers to the protein produced
from the obesity gene following transcription and deletions
of introns, translation to a protein and processing to the
mature protein with secretory signal peptide removed, e.g.,
from the N-terminal valine-proline to the C-terminal
cysteine of the ~ature protein. The ~ouse obesity protein
and human obesity protein are pub~ished in Zhang et al.
Nature 372: 425-32 (1994) The rat obesity protein is
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published in Murakami et al., R;oc~h~m;~l ~n-l Biol~hvsi~-Al
Res~r~ C-mm. 209(3): 944-52 (1995). The nur~ering of
amino acids in the present specification is consecutively
from the amino terminus of the full length, mature protein.
S Such uniform numbering of amino acid residues is well
accepted by those of ordinary skill in the art. In the
human, murine, porcine, bovine, and rat obesity proteins,
the cysteines associated with disul~ide formation are at
residues 96 and 146. The phrase "A is a polypeptide
consisting essentially o~ amino acid residues 1 to 95 of an
obesity protein" is intended to include the amino acids ~rom
the N-t~r~;nll.s to the ~irst cysteine associated with di-
sulfide bond formation. The phrase "B is a polypeptide
consisting essentially o~ amino acid residues 97 to 145 of
1~ the obesity protein" is intended to include the amino acids
from the ~irst cysteine to the C-t~rm;n~l cysteine of the
obesity protein. It is understood that the deletion of one
or more amino acids in a natural ~ariant or ~ragment as well
as any amino acid additions, including additions to the Cys
at position 146, that do not effect the novel and basic
characteristics o~ the invention are included in the
~f; n; tions of A and B. Renumbering the amino acid
residues o~ the protein is unnecessary and may result in
con~usion. For example, particularly with the murine and
hu~an obesity protein, a desGln(2~) variant has been
observed. Such variant is intended to be included in the
present invention.
Obesity protein analog refers to an obesity
protein having one or more amino acid substitutions,
preferably less than five and most pre~erably less than
three substitutions, and includes proteins disclosed of the
Formula ~III):
(SEQ ID NO: 1)
5 ~o 15
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
,
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Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
50 55 60
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
65 70 75 80
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg A~p Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
lO0 105 llO
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
115 l~0 125
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
145
Gly Cys (III)
wherein:
Xaa at position 28 is Gln or absent;
said protein having at least one of the following
substitutions:
Gln at position 4 is replaced with Glu;
Gln at position 7 is replaced with Glui
Asn at position 22 is replaced with Gln or Asp;
Thr at position 27 is replaced with Ala;
Xaa at position 28 is replaced with Glu;
Gln at position 34 is replaced with Glu;
Met at position 54 is replaced with methionine
sulfoxide, Leu, Ile, Val, Ala, or Glyi
Gln at position 56 is replaced with Glu;
Gln at position 62 is replaced with Glu;
Gln at position 63 is replaced with Glu;
Met at position 68 is replace~ with ~ethionine
sulfoxide, ~eu, Ile, Val, Ala, or Gly;
Asn at position 72 is replaced with Gln, Glu, or Asp;
Gln at position 75 is replaced with Glu;
Ser at position 77 is replaced with Ala;
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Asn at position 78 is replaced with Gln or Asp;
Asn at position 82 is replaced with Gln or Asp;
His at position 97 is replaced with Gln, Asn, Ala, Gly,
Ser, or Pro;
Trp at position 100 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu;
Ala at position 101 is replaced with Ser, Asn, Gly,
His, Pro, Thr, or Val;
Ser at position 102 is replaced with Arg;
Gly at position 103 is replaced with Ala;
Glu at position 105 is replaced with Gln;
Thr at position 106 is replaced with Lys or Ser;
Leu at position 107 is replaced with Pro;
. Asp at position 108 is replaced with Glu;
Gly at position 111 is replaced with Asp;
Gly at position 118 is replaced with Leu;
Gln at position 130 is replaced with Glu;
Gln at position 134 is replaced with Glu;
Met at position 136 is replaced with methionine
sulfoxide, Leu, Ile, Val, Ala, or Gly;
Trp at position 138 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu; or
Gln at position 139 is replaced with Glu
or a rh~r~ceutically acceptable salt thereof.
A properly folded obesity protein or analog thereof is the
protein in the conformation or tertiary structure resulting
in a biologically active protein useful in treating obesity
and those conditions associated with obesity such as
diabetes, cardiovascular disease and cancer.
Ob gene -- re~ers to any nucleic acid se~uence
that hybridizes, and is at Least 50 % homologous, pre~erably
70 % homologous, and ~ost preferably 80 % homologous to the
native ob gene se~uences disclosed by Zhang et al. Natll~e
372. 425-32 (1994) and Murakami et al., Biochemical and
R;~hysi~al Research Comm. ~Q~(3): 944-52 (1995). The ob
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gene product is expressed specifically in adipose tissue and
regulates energy h~l~n~-e.
Obesity protein inclusion bodies -- refers to
insoluble protein aggregates or cytoplasmic aggregates
cont~;n;ng, at least in part, the obesity protein to be
recovered. Similarly, inclusion bodies when preparing an
obesity protein analog refers to insoluble protein
aggregates or cytoplasmic aggregates cont~ln;ng, at least in
part, the obesity protein analog to be recovered.
Plasmid -- an extrachromosomal sel~-replicating
genetic element.
Reading frame -- the nucleotide sequence from
which translation occurs "read" in triplets by the
translational apparatus of tRNA, ribosomes and associated
factors, each triplet corresponding to a particular amino
acid R~ e each triplet is distinct and of the same
length, the coding sequence must be a multiple of three. A
base pair insertion or deletion (termed a frameshift
mutation) may result in two different proteins being coded
~or by the sa~e DNA segment. To insure against this, the
triplet codons corresponding to the desired polypeptide must
be aligned in multiples of three fro~ the initiation codon,
i.e. the correct '~reading frame" ~ust be maint~i n~,
R~mh;n~nt DNA Cloning Vector -- any autonomously
replicating agent including, but not limited to, plasmids
and phages, comprising a DNA ~olecule to which one or more
additional DNA sesments can or have been added.
Reco~binant DNA Expression Vector -- any
recombinant DNA cloning vector in which a promoter has been
incorporated.
Replicon -- A DNA sequence that controls and
allows for autono~ous replication of a plasmid or other
vector.
Rl and R2 are independently H or in conjunction
with the sulfur to which it is bound forms a mixed
disulfide; a ~ixed disul~ide is recognized and understood to
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be derive~ ~rom a thiol reducing reagent capable of
disul~ide exch~n~e. Suitable thiol reagents include one or
more mercapto reagents cont~; n; ng a free -SH that is capable
of ~orming a mixed disulfide and operable in disulfide
exch~n~e. Thiol therefore includes, but is not limited to,
cysteine, 2-mercaptoethanol, glutathionine, cyst~m; ne, ~-
mercaptoethanol (BME), and the like. Thiol reagents such as
dithiothreitol (DTT), dithioerythritol (DTE) are also
included in the present invention; but are less pre~erred
due to instability of the mixed disul~ide ~ormed with such
reagents. Accordingly, preferred moieties represented by R
or R2 include S03, SCH2CH(NH2)(COOH), SCH2CHNH2, SCH2CH20H,
and H2NCH(COOX)CX2CH2CONHCH(CH2S)CONHCH2COO-. Provided,
however, both Rl and R2 are not H.
Transcription -- the process whereby information
contained in a nucleotide se~uence o~ DNA is transferred to
a complementary RNA sequence.
Translation -- the process whereby the genetic
information of messenger RNA is used to specify and direct
the synthesis of a polypeptide chain.
Tris -- an abbreviation for tris(hydroxymethyl)-
amino~ethane.
Treating -- describes the management and care of a
patient for the purpose o~ co~bating the disease, condition,
or disorder a~d includes the ~; n; stration of a compound o~
present invention to prevent the onset of the symptoms or
co~plications, alleviating the symptoms or complications, or
~l;m;n~ting the disease, con~ition, or disorder. Treating
obesity therefore includes the inhibition of food intake,
the inhibition o~ weight gain, and inducing weight loss in
patients in need thereo~.
Vector -- a replicon used ~or the transfor~ation
of cells in gene ~anipulation bearing polynucleotide
se~uences corresponding to appropriate protein molecules
which, when co~bine~ with appropriate control sequences,
con~er speci~ic properties on the host cell to be
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transformed. Plasmids, viruses, an~ bacteriophage are
suitable vectors, since they are replicons in their own
right. Artificial vectors are constructed by cutting and
joining DNA molecules from di~ferent sources using
restriction enzymes and ligases. Vectors include
Recombinant DNA cloning vectors and Recombinant DNA
expression vectors.
The amino acids abbreviations are accepted by the
United States Patent and TrAA~k Of~ice as set forth in 37
C.F.R. 1.822 (b)(2) (1993). One skilled in the art would
recognize that certain amino acids are prone to
rearrangement. For example, Asp may rearrange to
aspartimide and isoasparigine as described in I. Sch~n et
al., Int. J. Pe~tide Protein Res. 14: 485-94 (1979) and
references cite~ therein. These rearrange~ent derivatives
are included within the scope of the present invention.
Unless otherwise indicated the amino acids are in the L
con~iguration.
As noted, the present in~ention provides a protein
of the Formula (I):
96
A - N - CH -
H2 C~
R
R2
HOOC - C- MX
146 (I)
wherein:
A is a polypeptide consisting essentiaLly of amino acid
residues 1 to 95 of an obesity protein or analog thereof;
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B is a polypeptide consisting essentially o~ amino acid
residues 97 to 145 of the protein;
Rl and R2 are independently H or in conjunction with
the sul~ur to which it is bound forms a mixed disul~ide;
provided that both Rl and R2 are not H.
In a pre~erred embodiment, A is a polypeptide of
the formula: (SEQ ID N0: 2)
s lo 15
R3 - Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys
20 25 30
Thr Ile Val Thr Arg Ile Asn Asp Ile Ser ~is Thr Xaa Ser Val Ser
35 40 45
Ser Lys Gln ~ys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro
Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln
65 70 75
Ile ~eu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn A~p
80 85 go 95
Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
wherein:
R3 is absent, Met, Met-R4, or a leader se~uence;
R4 is any a~ino acid except Pro;
Xaa at position 28 is Gln or absent;
Gln at position 4 is optionally replaced with Glu;
Gln at position 7 is optionally replaced with Glu;
Asn at position 22 is optionally replaced with Gln or
Asp;
Thr at position 27 is optionally replaced with Ala;
Xaa at position 28 is optionally replaced with Glu;
Gln at position 34 is optionally replaced with Glu;
Met at position 54 is optionally replaced with
methionine sul~oxide, Leu, Ile, Val, Ala, or Gly;
Gln at position 56 is optionally replaced with Glu;
Gln at position 62 is optionally replaced with Glu;
Gln at position 63 is optionally replaced with Glu;
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Met at position 68 is optionally replaced with
methionine sulfoxide, Leu, Ile, Val, Ala, or Gly;
As~ at position 72 is optionally replaced with Gln,
Glu, or Asp;
Gln at position 75 is optionally replaced with Glu;
Ser at position 77 is optionally replaced with Ala;
Asn at position 78 is optionally replaced with Gln or
Asp; or
Asn at position 82 is optionally replaced with Gln or
Asp.
Other preferred embodiments include those wherein A is
a polypeptide of the formula: ( SEQ ID NO: 2)
. 5 10 15
R3 - Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys
Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser
35 40 45
Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro
50 55 60
Ile Leu Thr Leu Ser Lys Met Asp Gln ~hr Leu Ala Val ~yr Gln Gln
65 70 75
Ile Leu Thr Ser Net Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp
80 85 90 95
Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
wherein:
Xaa at position 28 is Gln or absent.
Yet ad~itional preferred e~bodi~ents include
proteins wherein B is a polypeptide of the Forrnula:
(SEQ ID NO: 3)
~ 100 105 110
His Leu Pro Trp Ala Ser Gly Leu Glu ~hr Leu Asp Ser Leu Gly Gly
115 120 125
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu ~rp Gln Leu Asp Leu Ser Pro
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145
Gly ;
wherein:
His at position 97 is optionally replaced with Gln,
Asn, Ala, Gly, Ser, or Pro;
Trp at position 100 is optionally replaced with Ala,
Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val
or Leu;
Ala at position 101 is optionally replaced with Ser,
Asn, Gly, His, Pro, Thr, or Val;
Ser at position 102 is optionally replaced with Arg;
Gly at position 103 is optionally replaced with Ala;
Glu at position 105 is optionally replaced with Gln;
Thr at position 106 is optionally replaced with Lys or
Ser;
Leu at position 107 is optionally replaced with Pro;
Asp at position 108 is optionally replaced with Glu;
Gly at position 111 is optionally replaced with Asp;
Gly at position 118 is optionally replaced with Leu;
Gln at position 130 is optionally replaced with Glu;
Gln at position 134 is optionally replaced with Glu;
Met at position 136 is optionally replaced with
~ethionine sulfoxide, Leu, Ile, Val, Ala, or Gly;
Trp at position 138 is optionally replaced with Ala,
Glu, Asp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val
or Leu; or
Gln at position 139 is optionally replaced with
Glu.
Other preferred embodiments include proteins
wherein ~ is a polypeptide of the for~ula:
(SEQ ID NO:3)
100 105 110
His Leu Pr~ Trp Ala Ser Gly Leu Glu ~hr Leu Asp Ser Leu Gly Gly
115 120 125
Val Leu Glu Ala Ser Gly Tyr Ser ~hr Glu Val Val Ala Leu Ser Arg
130 135 140
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Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
145
Gly :
wherein:
Trp at position 100 is optionally replaced with Ala,
Glu, ~sp, Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val
or Leu.
One skilled in the art would recognize that in the
production o~ an obesity protein or analog thereof in a
procaryote expression system, it is necessary to express the
protein with a N-ter~inal extension appendaged to the N-
term;nllS o~ the protein. The N-t~rm;n~l extension is
optionally cleave~ from the protein prior to ~m; n; ~tration.
Pre~erred N-terminal extensions are Met or Met-R4- wherein
R4 is any amino acid except Pro.
The present invention also includes proteins
wherein A optionally includes one or ~ore amino acid leader
sequence that may be used for purification or other
purposes. Most preferred leader sequences include Gly-Ser-
His-~et lSEQ ID NO: 4), Met-Gly-Ser-Ser-His-His-His-His-His-
His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His-Met (SEQ ID NO:
5), Leu-Glu-Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO: 6), Glu-Ala-
Glu-Ala (SEQ ID NO: 7), Leu-Glu-Lys-Arg- (SEQ ID NO: 8),
Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-
Pro-Arg-Gly-Ser-Pro (SEQ ID NO: 9), and Gly-Ser-Pro- (SEQ ID
NO: 10). Such an N-t~rm;n~l extension and/or leader
sequence does not effect the basic an~ novel characteristics
of this invention.
The compounds of Form~ (I) are fol~ing
inte~ediates of the biologically active obesity protein.
Preferred obesity proteins are the native sequences and
analogs such as those described in Basinski et al., in U.S.
applications serial nu~ber 08/383,638, ~iled Fe~ruary 6,
1995, and serial nu~ber 08/588,061, filed January 19, 1996,
of the Form~ (Ilr). ~he ~urine and ~ovine seguences are
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described in Hansen M. Hsiung and Dennis P. Smith, in U.S.
application serial number 08/445,305, filed May 19, 1995
herein incorporated by re~erence. Most preferred proteins
o~ the present in~ention include proteins of Formula (III),
SEQ ID SEQ ID N0:11, SEQ ID NO:12, SEQ ID NO: 13 and SEQ ID
NO:14.
SEQ ID N0:11
1 . 5 10 15
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
20 25 30
Ile Val Thr Ars Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
35 40 45
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
65 70 75 80
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg Asp Leu heu His Val Leu Ala Phe Ser Lys Ser Cys
100 105 110
His Leu Pro Gln Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly
115 120 125
3 0 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu Gln Gln Leu Asp Leu Ser Pro
145
Gly Cys
wherein:
Xaa at position 28 is Gln or absent.
SEQ ID NO:12
1 5 10 15
Val Pro Ile Trp Arg Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
20 25 30 '
lle Val Thr Arg lle Ser AE;p Ile Ser His Met Gln Ser Val Ser Ser
35 40 45
Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val
50 55 60
Leu Ser Leu Ser Lys Net Asp Gln ~hr Leu Ala Ile Tyr Gln Gln Ile
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65 70 75 80
Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys
100 105 110
Pro Leu Pro Gln Ala Arg Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly
0 115 120 125
Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 1~0
Leu Gln Gly Ala Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro
145
Gly Cys
SEQ ID NO:13
2 0 1 5 10 15
Val Pro Ile Cys Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
20 25 30
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
35 40 45
~ys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Leu
50 55 60
Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile
Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ala Ser Lys Ser ~ys
100 105 110
Pro Leu Pro Gln Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val
115 120 125
Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro
145
Gly Cys
wherein Xaa at position 28 is Gln or absent.
Hurnan Obesitv Prc:~tein
SEQ ID NO :14
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l 5 lO 15
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
20 25 30
Ile Val Thr Arg Ile Asn A~p Ile Ser His Th~ Xaa Ser Val Ser Ser
35 40 45
Lys Gln Ly~ Val Thr Gly Leu Asp Phe Ile Pro Gly ~eu ~is Pro Ile
50 55 60
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
65 70 75 80
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
lO0 105 llO
His Leu Pro ~rp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
115 120 125
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
145
Gly Cys
wherein:
Xaa at position 28 is Gln or absent.
Other pre~erred proteins of the present invention
are those of Formula (III), wherein:
Gln at position 4 is replaced with Glui
Gln at position 7 is replaced with Glu;
Asn at position 22 is replaced with Gln or Asp;
Thr at position 27 is replaced with Ala;
Gln at position 28 is replaced with Glu;
Gln at position 34 is replaced with Glu;
Met at position 54 is replaced with ~ethionine
sulfoxide, Leu, or Ala;
Gln at position 56 is replacea with Glu;
Gln at position 62 is replaced with Glu;
Gln at position 63 is replaced with Glu;
Met at position 68 is replaced with methionine
sul~oxide, or Leu;
-
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Asn at position 72 is replaced with Gln or Asp;
Gln at position 75 is replaced with Glu;
Asn at position 78 is replaced with Gln or Asp;
Asn at position 82 is replaced with Gln or Asp;
Gln at position 130 is replaced with Glu;
Gln at position 134 is replaced with Glu;
Met at position 136 is replaced with methionine
sulfoxide, Leu, Ile; or
Gln at position 139 is replaced with Glu.
Other preferred proteins are those of Formula
(III) wherein:
Asn at position 22 is replaced with Gln or Asp;
Thr at position 27 is replaced with Ala;
~et at position 54 is replaced with methionine
sulfoxide, Leu, or Ala;
Met at position 68 is replaced with methionine
sul~oxide, or Leu;
Asn at position 72 is replaced with Gln or Asp;
Asn at position 78 is replaced with Gln or Asp;
Asn at position 8Z is replaced with Gln or Asp; or
Met at position 136 is replaced with methionine
sul~oxide, Leu, or Ile.
Still yet additional preferred proteins are those
o~ For~ula (III), wherein:
Asn at position 22 is replaced with Gln or Asp;
~hr at position 27 is replaced with Ala;
Met at position 54 is replaced with Leu, or Ala;
Met at position 68 is replaced with Leu;
Asn at position 72 is replaced with Gln or Asp;
Asn at position 78 is replaced with Gln or Asp;
Asn at position 82 is replaced with Gln or Asp; or
Met at position 136 is replaced with Leu, or Ile.
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Most significantly, other preferred embodiments
are specific substitutions to amino acid residues 97 to 111
and/or 138 of the proteins of SEQ ID NO: 1. These
substitutions result in significantly improved stability and
are superior therapeutic agents. These specific proteins
are more readily f~rm~ ted and are more pharmaceutically
elegant, which results in superior delivery of therapeutic
doses. Accordingly, preferred embodiments are proteins of
the For~ula (IV):
(SEQ ID NO:l)
5 lo 15
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
20 25 30
Ile Val ~hr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
~o 45
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
so 55 60
Leu Thr Leu Ser Lys Met A~p Gln Thr Leu Ala Val Tyr Gln Gln Ile
65 70 75 80
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 so 95
G~u Asn Leu Arg Asp Leu Leu Hi~ Val Leu Ala Phe Ser Lys Ser Cys
lOo 105 llo
3 0 Hi s Leu Pr~ Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
115 120 125
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Net Leu Trp Gln Leu Asp Leu Ser Pro
145
Gly Cys ( IV)
wherein:
Xaa at position 28 is Gln or absent;
said protein having at least one substitution selected from
the group consisting of:
His at position 97 is replaced with Gln, Asn, Ala, Gly,
Ser, or Pro;
Trp at position 100 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu;
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Ala at position 101 is replaced with Ser, Asn, Gly,
His, Pro, Thr, or Val;
Ser at position 102 is replaced with Arg;
Gly at position 103 is replaced with Ala;
Glu at position 105 is replaced with Gln;
Thr at position 106 is replaced with Lys or Ser;
Leu at position 107 is replaced with Pro;
Asp at position 108 is replaced with Glu;
Gly at position 111 is replaced with Asp; or
Trp at position 138 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu;
or a pharmaceutically acceptable salt thereof.
Preferred proteins are proteins of the Form
(V):
(SEQ ID N0:15)
5 10 15
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
20 25 30
Ile Val Thr Arg Ile Asn A~p Ile Ser His Thr Gln Ser Val Ser Ser
g5
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu ~is Pro Ile
50 55 60
Leu Thr Leu Ser Lys Net Asp Gl~ Thr Leu Ala Val Tyr Gln Gln Ile
65 70 75 80
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg Asp Leu Leu His Val ~eu Ala Phe Ser Lys Ser Cys
100 105 110
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
115 120 125
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
145
Gly Cys (V)
said protein having at least one substitution selected ~rom
the group consisting o~:
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His at position 97 is replaced with Gln, Asn, Ala, Gly,
Ser, or Pro;
Trp at position 100 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu;
Ala at position 101 is replaced with Ser, Asn, Gly,
His, Pro, Thr, or Val;
Ser at position 102 is replaced with Arg;
Gly at position 103 is replaced with Ala;
Glu at position 105 is replaced with Gln;
Thr at position 106 is replaced with Lys or Ser;
Leu at position 107 is replaced with Pro;
Asp at position 108 is replaced with Glu;
Gly at position 111 is replaced with Asp; or
Trp at position 138 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu;
or a p~r~ceutically acceptable salt thereof.
More preferred proteins of the Formula (V) are
those wherein:
His at position 97 is replaced with Gln, Asn, Ala, Gly,
Ser or Pro;
Trp at position 100 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln or Leu;
Ala at position 101 is replaced with Ser, Asn, Gly,
His, Pro, Thr or Val;
Glu at position 105 is replaced with Gln;
Thr at position 106 is replaced with Lys or Ser;
Leu at position 107 is replaced with Pro;
Asp at position 108 is replaced with Glu;
Gly at position 111 is replaced with Asp; or
Trp at position 138 is replaced with Ala, Glu, Asp,
Asn, Met, Ile, Phe, Tyr, Ser, Thr, Gly, Gln, Val or Leu.
Other pre~erred proteins of the Formula (V) are
those wherein:
His at position 97 is replaced with Ser or Pro;
~rp at position 100 is replaced with Ala, Gly, Gln,
Val, Ile, or Leu;
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Ala at position 101 is replaced with l~r; or
Trp at position 138 is replaced with Ala, Ile, Gly,
Gln, Val or Leu.
Yet still additional preferred proteins of the
5 Form~ (V) are those wherein:
His at position 97 is replaced with Ser or Pro;
Trp at position 100 is replaced with Ala, Gln or Leu;
Ala at position 101 is replaced with Thr; or
Trp at position 138 is replaced with Gln.
Most preferred species of Formula (V) include
species o~ SEQ ID NO: 16 and 17:
(SEQ ID NO:16)
5 lo 15
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
35 40 45
Lys Gln Lys Val Thr Gly ~eu Asp Phe Ile Pro Gly Leu His Pro Ile
50 55 60
Leu Thr Leu Ser Lys Net Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
65 70 75 80
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
85 90 95
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
100 105 110
His Leu Pro Ala Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
115 120 125
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 135 140
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
145
Gly Cys
(SEQ ID NO:17)
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
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Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
100 105 110
Hls Leu Pro Gln Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
115 120 125
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
130 ~35 140
Leu Gln Gly Ser ~eu Gln A~p Met Leu Trp Gln ~eu Asp Leu Ser Pro
145
Gly Cys
Most significantly, the present invention provides
an int~rme~;~te, which leads to a very ef~icient conversion
to the biologically active protein conformation of the
obesity protein. There~ore, by preparing the int~r~ te
of the present invention, the teriary structure of the
biologically active protein is formed first. Thus, allowing
almost quantitative conversion of the disulfide bonds. The
formation o~ S-S linked dimer or other multimers is
m; n;rn;zed.
The protein of the present invention is prepared
by recombinant DNA technology or well known chemical
procedures, such as solution or solid-phase peptide
synthesis, or semi-synthesis in solution beg;nn;ng with
protein fragments coupled through conventional solution
~ethods. Preferably, a protein of Form~ (III), or SEQ ID
N~s:ll, 12, 13, or 14 is prepared by recombinant synthesis.
Reco~binant methods are preferred if a high yield
is desired. The basic steps in the recombinant production
of protein include:
-
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a) construction of a synthetic or semi-synthetic
(or isolation from natural sources) DNA
encoding the protein,
b) integrating the coding sequence into an
expression vector in a manner suitable for
the expression o~ the protein either alone or
as a fusion protein,
c) transform; n~ an appropriate prokaryotic host
cell with the expression vector, and
d) recovering and puri~ying the recombinantly
produced protein.
a. Gene Construction
Synthetic genes, the n vitro or n vivo
transcription and translation of which will result in the
production of the protein may be constructed by techniques
well known in the art. Owing to the natural degeneracy o~
the genetic code, the skilled artisan will recognize that a
sizable yet definite nl~h~ of DNA sequences may be
constructed which encode the proteins. In the preferred
practice of the invention, synthesis is achieved by
recombinant DNA technology.
Methodology of synthetic gene construction is well
known in the art. For example, see Brown, et al. (1979)
Methods in Enzymology, ~e~ic Press, N.Y., Vol. 68, pgs.
109-151. The DNA sequence corresponding to the synthetic
protein gene may be generated using conventional DNA
synthesizing apparatus such as the Applied Biosystems Model
380A or 380B DNA synthesizers (commercially available from
Applied Biosyste~s, Inc., 850 Lincoln Center Drive, Foster
City, CA 94404).
It may desirable in some applications to modify
the coding sequence of the protein so as to incorporate a
convenient protease sensitive cleavage site, e.g., between
the signal peptide and the structural protein facilitating
the controlled excision of the signal peptide from the
~usion protein construct. Techni~ues for making
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substitutional mutations at predetermined sites in DNA
having a known sequence are well known, for example M13
primer mutagenesis. The mutations that might be made in the
DNA encoding the present anti-obesity proteins must not
place the sequence out of reading frame and preferably will
not create complementary regions that could produce
secondary mRNA structure. See DeBoer et al., EP 75,44~A
(1983).
The gene encoding the protein may also be created
by using polymerase chain reaction (PCR). The template can
be a cDNA library (commercially available ~rom CLO~ ~ or
STRATAGENE) or mRNA isolated from human adipose tissue.
Such methodologies are well known in the art Maniatis, et
al. Molecular Cloninq: A Laboratorv M~nl~ 1, Cold Spring
Harbor Press, Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York (1989).
b. Direct ex~ression or Fusion ~rotein
The obesity proteins may be made either by direct
expression or as fusion protein comprising the protein
followed by enzymatic or chemical cleavage. A variety of
peptidases (e.g. trypsin) which cleave a polypeptide at
specific sites or digest the peptides from the amino (e.g.
~i~m;nopeptidase) or carboxy termini of the peptide chain
are known. Furth~rm~re~ particular chemicals (e.g. cyanogen
bromide) will cleave a polypeptide chain at specific sites.
The skilled artisan will appreciate the modifications
necessary to the amino acid sequence (and synthetic or semi-
synthetic co~ing sequence if recombinant means are employed)
to incorporate site-specific internal cleavage sites.
e.g., Carter P., Site Specific Proteolysis of Fusion
Proteins, Ch. 13 in Protein Purification: From Molecular
~h~n; s~s to r.~r~e Scale Processes, American Chemical Soc.,
W~ h; n~ton, D. C . ( 1990 ) .
c. Vector Con~truction
Construction of suitable vectors cont~;n;n~ the
desired coding and control sequences employ st~n~d
-
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ligation techni~ues. Isolated plasmids or DNA fragments are
diges~ed by restriction enzymes, tailored, and religated to
form the plasmids reguired.
To ef~ect the translation of the desired protein,
one inserts the engineered synthetic DNA sequence in any of
a plethora of appropriate recombinant DNA expression vectors
through the use of appropriate restriction endonucleases. A
synthetic coding sequence is designed to possess restriction
~n~onl~clease cleavage sites at either end of the transcript
to facilitate isolation from and integration into these
expression and amplification plasmids. The isolated cDNA
coding sequence may be readily modified by the use o~
synthetic linkers to facilitate the incorporation of this
sequence into the desired cloning vectors by techniques well
known in the art. The particular ~n~on~lrleases employed
will be dictated by the restrict~on ~n~onllclease cleavage
pattern of the parent expression vector to be employed. The
choice of restriction sites are chosen so as to properly
orient the coding sequence with control sequences to achieve
proper in-frame reading and expression of the protein.
In general, plasmid vectors are used which contain
promoters and control se~uences which are derived from
species compatible with the host cell. The vector
ordinarily carries a replication site as well as marker
sequences which 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 (Bolivar, et al., Gene 2: 95 (1977)).
Plasmid pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR322 plasmid, or other
~icrobial plasmid must also contain or be modified to
contain promoters and other control elements commo~ ly used
in rec~mh;n~nt DNA t~chnology.
The desired coding seguence is inserted into an
expression vector in the proper orientation to be
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transcribed and translated from a promoter and ribosome
h~n~;ng site, both of which should be functional in the host
cell. An example of such an expression vector is a plasmid
described in Belagaje et al., U.S. patent No. 5,304,493, the
t~h;n~s o~ which are herein incorporated by reference.
The gene encoding A-C-B proinsulin described in U.S. patent
No. 5,304,493 can be removed from the plasmid pRB182 with
restriction enzymes NdeI and ~HI. The genes encoding the
protein of the present invention can be inserted into the
plasmid backbone on a ~ HI restriction fragment
cassette.
~, Procarvotic ex~xession
In general, procaryotes are used for cloning of
DNA sequences in constructing the vectors useful in the
invention. For example, E. coli K12 strain 294 (ATCC No.
31446) is particularly useful. Other microbial strains which
may be used include E. coli B and ~. coli X1776 (ATCC No.
31537). These examples are illustrative rather than
limiting.
Also, prokaryotes are used for expression of
recombinant proteins. The aforementioned strains, as well as
E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such
as Racillus subtilis, and other enterobacteriaceae such as
!~almonell~ tv~h;m~ m or Serratia marces~n~ and various
pseu~ n~ species may be used. Promoters suitable for use
with prokaryotic hosts include the ~-lactamase (vector
pGX2907 [ATCC 39344] contains the replicon and ~-lactamase
gene) and lactose promoter systems (Chang et al., Na~ure,
275:615 (1978); and Goeddel et al., Nature ~ 544 (1979)),
alkaline phosphatase, the tryptophan (trp) promoter system
(vector pATHl [ATCC 37695] is designed to facilitate
expression of an open reading frame as a trpE fusion protein
under control o~ the trp promoter) and hybrid promoters such
as the tac promoter (isolatable from plasmid pDR540 ATCC-
37282). However, other functional bacterial promoters,
whose nucleotide sequences are generally known, enable one
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of skill in the art to ligate them to DNA encoding the
protein using linkers or adaptors to supply any required
restriction sites. Promoters for use in bacterial systems
also will contain a Shine-Dalgarno sequence operably linked
to the DNA encoding protein.
The ~ollowing preparations are presented to further
illustrate the preparation of the proteins described herein.
Pre~aration 1
A DNA sequence encoding the protein of SEQ ID NO:14
with a Met-Arg N-t~rm;n~l extension was obtained using
standard PCR methodology. A forward primer (5'-GG GG CAT
ATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC, SEQ ID
NO:18) and a reverse primer (5'-GG GG GGATC CTA TTA GCA CCC
GGG AGA CAG GTC CAG CTG CCA CAA CAT, SEQ ID NO:l9) was used
to amplify sequences from a human ~at cell library
(commercially available from CLO~l~). The PC~ product is
cloned into PCR-Script (available from STRATAGENE) and
sequenced.
Pre~aration 2
Vector Construction
A plasmid cont~;n;n~ the DNA sequence encoding the
desired protein is constructed to include NdeI and ~HI
restriction sites. The plasmid carrying the cloned PCR
product is digested with NdeI and ~mHI restriction enzymes.
The small ~ 450bp ~ragment is gel-purified and ligated into
the vector pRB~82 ~rom which the coding sequence for A-C-B
proinsulin is deleted. The ligation products are
trans~ormed into E. coli DHlOB (commercially available ~rom
GIBCO-BRL) and colonies growing on tryptone-yeast (DIFCO)
plates supplemented with 10 ~g/mL of tetracycline are
analyzed. Plasmid DNA is isolated, digested with NdeI and
HI and the resulting fragments are separated by agarose
gel electrophoresis. Plasmids cont~;n;ng the expected ~
450bp NdeI to ~8~HI fragment are kept. E. coli B BL21
(DE3) (commercially available fro~ NOVOGEN) are trans~ormed
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with this second plasmid expression suitable for culture ~or
protein production.
The techniques of transforming cells with the
a~orementioned vectors are well known in the art and may be
found in such general references as Maniatis, et al. (1988)
Molec~ ~ Cloning: A Laboratorv Manual, Cold Spring Harbor
Press, Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York or Cl~rrent Protocols in Molecular Bioloqv (1989)
and supplements. The techniques involved in the
transformation of E. coli cells used in the preferred
practice of the invention as exemplified herein are well
known in the art. The precise conditions under which the
transformed F.. coli cells are cultured is dependent on the
nature of the E. coli host cell line and the expression or
cloning vectors employed. For example, vectors which
incorporate thermoinducible promoter-operator regions, such
as the c1857 thermoinducible l~h~-phage promoter-operator
region, require a temperature shift ~rom about 30 to about
40 degrees C. in the culture conditions so as to induce
protein synthesis.
In the preferred embodiment o~ the invention E.
coli K12 RV308 cells are e~ployed as host cells but numerous
other cell lines are available such as, but not limited to,
E. ÇQli K12 L201, L687, L693, L507, L640, L641, L695, L814
(~- ÇQli B). The trans~ormed host cells are then plated on
d~Liate media under the selective pressure of the
antibiotic correspon~; n~ to the resistance gene present on
the expression plasmid. The cultures are then incubated ~or
a time and temperature appropriate to the host cell line
employed.
When expressed in a high-level bacterial
expression syste~s, the protein often aggregate in granules
or inclusion bodies which contain high levels o~ the
overexpressed protein. Kreuger et al., in Protei~ Fol~; n~,
Gierasch and King, eds., pgs 136-142 (1990), American
Association ~or the Advancement of Science Publication No.
CA 02224867 1997-12-17
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-31-
89-18S, W~h;n~ton, D.C. The inclusion bodies comprising
the obesity protein are solubilized in a denaturant at a
concentration sufficient to solubilize the protein,
pre~erably in about 6 to about 8 M urea at a pH from about
pH 7 to about pH 12, more preferably 8 to 12. Most
preferably, the inclusion bodies are solubilized in about 6
to 7 M urea at a pH of about 8 to 10. The desired protein
concentration is about 0.1 mg/mL to the solubility o~ the
protein in the solution; more preferably 0.1 to 50 mg/mL and
most preferably 0.5 mg/mL to 5.0 mg/mL. Under these
conditions the protein is denatured.
Denatured protein molecules regain their native
conformation when the renaturation is carried out under
carefully controlled conditions. However, renaturation into
the biologically active conformation is not a quantitative
process; a multitude of non-~unctional species and
conformations of the molecule may be formed. The present
invention provides a key int~rm~;ate in the renaturation.
The cl~;m~ int~rme~;~te is prepared by adding
about 1 to 100 mM, pre~erably 1 to 20 mM and most preferably
1 to 10 mM, of a thiol reducing reagent containing a free
-SH that is operable in a disulfide interchange, preferably,
cysteine, cyst~m;ne, BME and the like. Preferred thiol
reagents are cysteine and cysteamine. The claimed
int~rmP~;~te forms in about 1 minute to 24 hours. The
int~rm~ te can be purified by filtration, chromatography
or other conventional methodology. Because the int~rme~;~te
is in the correct tertiary structure (native con~ormation),
the int~rme~;ate can be used as a biologically active
therapeutic agent and may offer advantages in efficacy or
onset of action. However, the intermediate is pre~erably
converted to the biologically active proteins of Formula
III.
Most unexpectedly, conversion of the int~rm~;ate
to the biologically active protein is highly efficient with
little or no precipitation of aggregated protein and with
CA 02224867 l997-l2-l7
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-32-
m;n;m~l formation of covalent dimers or higher order
multimers. Protecting the cysteine residues of the protein
by the formation of mixed disulfides and thus forming the
int~rm~A;~te, allows for the formation of the tertiary
structure largely independent of disulfide bond formation.
This condition favors the intramolecular disulfide bond
formation in the monomeric biologically active protein.
The int~rm~A;ate is converted to the biologically active
protein of the Formula II by reducing the concentration of
the thiol reducing reagent and denaturant concentration of
the solution to effect disulfide bond formation. The
reduction of thiol and denaturant may be carried by
dilution, dialysis, diafiltration, or other techniques
appreciated in the art. Preferably, after solubilization of
the inclusion bodies and formation of the interme~;~te, the
solution is diluted to a protein concentration about 0.05
mg/mL to about 5.0 mg/mL and about 1 to 20 mM thiol and then
dialyzed or diafiltered. The bu~fer used for dialysis or
diafiltration is preferably PBS (phosphate buffered saline
with about 5 to about 10 mM phosphate and 50 to 500 mM NaCl)
at a pH of about 7.0 to 12.0 and more preferably 7.5 to 9Ø
Other suitable buffers include, but are not limited to, 4-
(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES),
or tris(hydroxymethyl)~m;n~m~thane (TRIS). Preferably, the
thiol reducing reagent and denaturant concentration is
reduced by diafiltration or dialysis against PBS or about 5
to 10 mM TRIS.
The conversion from the int~rmeA;ate to the
biologically active conformation is most remarkable --
approaching quantitative conversion. Thus, by passing
through the int~rm~ te, the biologically active protein of
the Formula II is produced in high yield. The formation of
the intPrm~A;ate also allows the fold to be conducted at
higher protein concentrations than a fold carried out
directly ~ro~ the ~ree -SH. The preferred range includes
O.05 to 5 mg/mL, preferably 0.1 to 3 mg/mL, and most
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-33-
preferably 1.0 to 2 mg/mL. A higher concentration during
the fold translates into lower volumes (smaller tanks) and
less downstream processing. The fold may be carried out in
the absence of glycerol or other agent added to prevent
protein aggregation. The ability to fold at large scale in
the absence of such agents is significant because such
agents, particularly glycerol, must be removed in downstream
purification.
Thus, by processing through the int~rm~ te, the
present invention further provides an efficient process of
preparing proteins of the Formula II:
(a) Solubilizing obesity protein inclusion bodies
in a solution comprising:
a denaturant at a concentration sufficient to
solubilize the protein; and
a thiol reducing reagent at a concentration of 1
to 100 mM at a pH from about 7 to about 12;
(b) Reducing the thiol and denaturant
concentration of the solution to effect disulfide bond
formation.
Preferably, inclusion bodies are solubilized by
the addition of about 6 to 8 M urea and about 3 to 7 mM
cysteine in a 8 to 12 mM Tris buffer at about pH 8 to 12 and
more preferably at a pH of about 8 to 10. Under these
conditions the mixed disulfide intermediate forms and is
optionally purified by filtration and/or chromatography.
Significantly, the efficiency of the formation of ~he single
intra-chain disul~ide from the intermediate is increased by
the ~; n~ additional thiol reducing reagent prior to
dilution, diafiltration or dialysis. Preferably, thiol is
added so that it is in molar excess -- preferably 1 to 6000
fold, more preferably 3 to 6000 fold, excess. Most
preferably, 5 mM thiol, preferably cysteine, is added.
Once converted to the biologically active protein,
the protein is purified from the reaction mixture by
techniques appreciated in the art such as ion exchange
CA 02224867 1997-12-17
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-34-
chromatography, size exclusion chromatography, reverse phase
chromatography, and the like.
The int~rm~late of Formula (I) is stable in the
presence or absence of denaturant. The intermediate is
soluble in PBS suggesting proper tertiary structure
formation. If desired the intermediate may be purified by
technigues known in the art and including size exclusion,
ion exchange, reversed phase chromatography. The
intermediate was characterized on HPLC. A chromatogram for
representative int~rme~;~tes of the cl~;me~ invention are
presented in Figure 1. Furthermore, the interm~;Ate is
identified on SDS-PAGE (sodium dodecyl-sulphate-
polyacrylamide gel electrophoresis as a slower migrating
species than the biologically active protein.
The following examples are presented to further
illustrate the invention described herein. The scope of the
present invention is not to be construed as merely
consisting of the following examples.
Com~arative Example 1
Protein of SEQ ID NO:14 wherein Xaa at position 28
is Gln and having a Met-Arg N-t~rm; n~ 1 extension was
produced as granules (inclusion bodies). The granules were
isolated by a st~n~Ard procedure using differential
centrifugation. These granules were solubilized in 6 M
guanidine-HC1, 10 mM sodium acetate (pH 5.0), 1 mM DTT for 1
hour at room temperature. The mixed disulfide of the
present invention was not detectable under these conditions.
The cysteine of the obesity protein is protonated under
these conditions to form (-SH). Solubilized protein was
clarified by centrifugation and renaturation o~ the
clarified protein solution was initiated by dilution (fold
dilution = 1:1500) into 20 % glycerol, 5 mM sodium acetate
(pH 5.0), 5 mM CaCl2 to a final protein concentration of
0.025 mg/mL with thorough mixing. The solution became hazy
and protein aggregation was noted shortly a~ter dilution.
The dilute protein solution was allowed to stand without
CA 02224867 1997-12-17
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-35-
m;x;n~ at room temperature for 8 hours, and the pH of the
solution was raised to 8.68 by addition of solid Tris base
to 10 mM. The solution was clarified by centrifugation and
analyzed by SDS-PAGE under non-reducing conditions, reverse
phase HPLC and ESI-mass spectroscopy. Analysis indicated an
overall recovery of 68 % of the protein o~ which 7 % was
covalent dimer yielding a 63 % recovery of monomeric protein
and a 32 % loss of protein.
Com~arative ~xa~le 2
Protein of SEQ ID NO:14 wherein Xaa at position 28
is Gln and having a Met-Arg N-~rm; n~l extension was
produced as granules (inclusion bodies). The granules were
isolated by a st~n~rd procedure using di~er~nt;~l
centrifugation. These granules were solubilized in 6 M
guanidine-HCl, 10 ~M sodium acetate (pH 4.5), 1 ~M DTT at a
protein concentration of 1.3 mg/~L ~or 1 hour at room
temperature. The mixed disul~ide o~ the present invention
was not detectable under these con~itions. The cysteine is
protonated under these conditions to form (-SH).
Solubilized protein was clari~ied by
centrifugation and renaturation o~ the clarified protein
solution was initiated by dilution (fold dilution = 1:55)
into 20 ~ glycerol, 20 ~M Tris (pH 8.4), 2.5 mM CaCl2 to a
final protein concentration o~ 0.025 mg/mL with thorough
mixing. The dilute protein solution was allowed to stand
without mixing at room temperature for 18 hours. The
solution was clarified by centrifugation and analyzed by
SDS-PAGE under non-reducing conditions, reverse phase HPLC
an~ ESI-~ass spectroscopy. Analysis indicated a recovery o~
>95 ~ of the protein of which 16 % was covalent dimer
yielding a recovery of monomeric protein of 80 ~.
ExamDle 1
Protein o~ SEQ ID NO:14 wherein Xaa at position 28
is Gln and having a Met-Arg N-t~r~i n~l extension was
produced as granules (inclusion bodies). The granules were
isolated by a standard procedure using dif~er~nt;~l
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-36-
centrifugation. These granules were solubilized in 8 M
urea, 10 mM Tris (pH 8.0), 5 mM cysteine at a protein
concentration o~ 0.1 mg/~. Renaturation of the protein
solution was initiated by dialysis against PBS to remove
excess denaturant and cysteine. The solution was clarified
by centrifugation and analyzed by SDS-PAGE under non-
reducing conditions, reverse phase XPLC and ESI-mass
spectroscopy. Analysis indicated a recovery of ~95 % of the
protein of which ~ 1 ~ was covalent dimer yielding an~0 overall recovery of monomeric protein of 94 ~.
~nle 2
Protein o~ SEQ ID NO:ll wherein Xaa at position 28
is Gln with a Met-Asp N-term;n~l extension was produce~ as
granules (inclusion bodies). The granules were isolated by
a st~ n~ rd procedure using differential centrifugation.
These granules constituted the starting material ~or f'urther
purification. The granules were suspended in bu~er A (8 M
urea, 10 mM Tris (pH 8.0), 5 mM cysteine) and found to be
soluble in this buffer at high concentrations (up to 40 mg
protein/mL). Solubilized protein was clarif'ied either by
centrifugation or by filtration. The protein migrated as a
doublet band on nonreducing SDS-PAGE gels an~ as a single
band on reducing SDS-PAGE gels. This is due to the presence
of some protein with an int~rn~l disul~ide bond and some
protein with the cysteine residues present as mixed
disulfides with the cysteine ~rom.. the buffer. The protein
was initially purified by DEAE anion exchange chromatography
in the presence of bu~f'er A. Protein bound to the DEAE
resin was eluted with a NaCl gradient to 0.250 mM.
Nonr~nc;n~ SDS-PAGE analysis of fractions indicated that
most of the cont~m;n~ting proteins were present in the
l~A; n~ edge of the main Ob peak. Conservative pooling oi~
the DEAE ~ractions resuLted in relatively pure Ob protein
for r~n~tll~ation. Re~olding of the protein was initiated by
dilution o~ the protein to 0.1 mg/~L in PBS.
,
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-37-
The example was repeated by dilution into buffer A
and removing the denaturant and thiol by dialysis into PBS.
The protein r~m~; neA soluble a~ter the dialysis and migrated
as a single band on nonreducing SDS-PAGE. Reduction of the
protein resulted in a single band with slightly slower
mobility on SDS-PAGE indicating that the disulfide bond was
completely ~or~ed during the renaturation process. Final
puri~ication of the protein was achieved by size exclusion
chromatography (Superdex 75 column in PBS) purified Ob
protein ~igrated as a single band on SDS-PAGE, yielded a
single N-ter~; n~l amino acid sequence, which was con~irmed
by ESI-mass spec.
Exam~le 3
A protein o~ SEQ ID NO :12 was prepared in a m~nn~r
analogous to Example 1.
~xample 4
A protein of SEQ ID NO :13 may be prepared in a
manner analogous to Example 1.
E~nle 5
Incubation of the partially puri~ied SEQ ID NO:14
with a Met-Asp N-t~rm;n~l extension in 8 M urea, 10 mM tris,
5 mM cysteine pH 8.0 for 48 hours at 4~ C resulted in a
mixture of for~s of the protein. Nonreducing SDS-PAGE
indicated that d~uximately half of the protein had an
internal disulfide bond while the other half did not.
Analysis of the protein solution with the sulfhydryl reagent
DINB indicated that no free sulfhydryl was present in
solution. This indicates that the cysteine in solution had
for~ed mixed disul~ides with the cysteine in the Ob protein.
Thus the intermediate in the folding of the biologically
active Ob protein had been trapped.
An aliquot o~ this solution was diluted to 0.1
mg/mL in PBS. A second aliquot was diluted to 0.1 mg/mL in
PBS with 20 ~M DT~. Both diluted sa~ples were then dialyzed
a~;n~t a ~0,000 fold excess of PBS for 24 hours. Following
dialysis the samples were analyzed by nonr~lc;n~ SDS-PAGE.
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-38-
The sample diluted into PBS cont~;n~ both Ob protein with
the internal disulfide bond and Ob protein without the
internal disulfide (soluble int~rm~;~te). The sample
diluted into PBS/DTT contained only Ob protein with the
internal disulfide bond.
Once formed, the mixed disulfide between Ob and
cysteine is stable even a~ter removal of denaturant. The
mixed disulfide Ob protein is soluble in PBS suggesting
proper secondary structure ~ormation. Addition of excess
thiol regent st;m~ tes disulf'ide ex~h~nge and gradual
removal of the thiol regent f'avors ~ormation of the Ob
molecule with the int~rn~l disul~ide bond.
~n le 6
Protein of For~~ (III) (SEQ ID NO:l) wherein Xaa
at position 28 is Gln and Trp at position 100 is replaced
with Glu and having a Met-Arg N-terminal extension was
produced as granules (inclusion bodies). The granules were
isolated by a st~n~l~rd procedure using differential
centrif~'ugation. These granules constituted the starting
material ~or ~urther puri~ication. The inclusion bodies
were solubilized in 8 M urea, 10 mM Tris (pH 8.0), 5 mM
cysteine; and the ~ixed disulfide protein was puri~ied by
anion e~h~n~e chromatography. Purified protein was
incubated in Buf~er A for 48 hours at 4 C. C~nf; rm~tion
that the cysteine had formed a mixed disul~ide (Rl is
SCH2C(COOH)(NH2)), was carried out using 10 mM DTNB in 0.1 M
~ris at pH 8 (measuring the absence of ~ree -SH).
Renaturation of the solubilized protein was initiated by
dilution of the protein to 0.1 mg/mL with 8 M urea, 10 mM
Tris at pX 8.0 and dialysis initiated against PBS. After 24
hours, a 1 mL sample was eluted over a C18 HPLC at 1
mL/minute with a 30-70% ~H3CN 1;~ gra~ient. TWo peaks
were collected that ~igrated as two separate bands on SDS
PAGE gel in non-r~Al~;n~ bu~er. ~he early eluting peak
migrated slower on SDS PAGE, and the ~ass expected for the
claimed mixed disulfide ~nt~; n; n~ intermediates was
CA 02224867 1997-12-17
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-39-
con~irmed by Esr ~ass spectroscopy. The later eluting peak
migrated ~aster on SDS PAGE. The mass expected for intra-
disul~ide cont~; n; ng protein of Formula II was con~irmed.
The analysis con~irmed that protein formed the mixed
disul~ide inter,~ediate o~ the present invention prior to
conversion to the disulfide cont~;n;n~ protein.
~xamDle 7
Protein o~ For~ula (III) (SEQ ID NO:l) wherein Xaa
at position 28 is Gln and Trp at position 100 is replaced
with Gln and having a Met-Arg N-t~rmin~l extension was
produced as granules (inclusion bodies). The granules were
isolated by a standard procedure using di~erential
centrifugation. These granules constituted the starting
material for further purification. The granuLes were
dissolved in 8 M urea, 10 mM Tris (pH 8.0), 5 mM cysteine.
Renaturation of the protein was initiated by dilution of the
protein to 0.1 mg/mL and dialysis against PBS to remove
excess denaturant and cysteine.
~xam~le 8
Protein of For~ula (III) (SEQ ID NO:l) wherein Xaa
at position 28 is Gln and Trp at position 100 is replaced
with Ala and having a Met-Arg N-terminus extension was
produced as granules (inclusion bodies). The granules were
isolated by a st~n~d procedure using differential
centrifugation. These granules constituted the starting
material ~or ~urther puri~ication. The granules were
suspended in 8 M urea, 10 m~ Tris (pH 8.0), 5 ~M cysteine.
Renaturation o~ the protein was initiated by dilution o~ the
protein to 0.1 ~g/~L and dialysis against PBS to remove
excess denaturant and cysteine.
N-t~rm;n~- Met-Arg dipeptide is removed by the
using dipeptidyLa~inopeptidase (dDAP) by t~hni~ues
appreciate~ in the art. The protein puri~ied by cation
e~hAnge chro~atography.
Exam~le 9
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-40-
Protein of F~r~lllA (III) (SEQ ID NO:l) wherein Xaa
at position 28 is Gln ana Trp at position 100 is replaced
with Ala and having a Met-Arg N-terminal extension was
produced as granules (inclusion bodies). The granules were
isolated by a st~n~Ard procedure using differential
centri~ugation. These granules constituted the starting
material ~or ~urther puri~ication. The granules were
solubilized by the addition of 7 M urea and 5 ~M cysteine in
a 10 mM Tris bu~fer at around pH 8. The solubilized
preparation was then clarified by filtration. Under these
conditions the cysteine residues of the protein are present
as a co~bination of reduced and cysteinyl mixed disul~ides.
~he urea solubilized mixed disulfide int~rme~;Ate was
puri~ied by anion exchange chromatography on a Big Bead Q-
Sepharose column (Pharmacia Fine Chemicals) in 7 M urea, 10
mM ~ris, 5 mM cysteine at approximately pH 8. The product
is eluted by a linear gradient in NaCl. Additional cysteine
is added to the anion ~rch~nge puriEied intermediate, and
the pH adjusted to approximately pH 9. ~he intermediate is
then diluted to app~oximately 2 mg/mL with 7 M urea.
Folding and fo~mation of the single intra-chain disul~ide
bond is accomplished by the removal o~ urea and cysteine
using me~brane ultrafiltration/dia~iltration against 10 mM
Tris at around pH 9. Ultra~iltration ~embrane with a
n~mln~l ~olecular weight cut-o~ o~ 10,000 daltons were
used. ~he N-t~r~;nAl Met-Arg dipeptide is removed by the
using dipeptidyl~m;nopeptidase (dDAP) by techni~ues
appreciated in the art.
~he protein is A~m;nistered in a dose between
about 1 and 1000 ~g/kg. A pre~erred dose is fro~ about 10 to
100 ~g/kg o~ active co~pound. A typical daily dose for an
adult human is ~ro~ about 0.~ to 100 mg. In practicing this
method, co~pounds o~ the Formula (I) can be ~;n;~tered in
a single daily aose or i~ multiple doses per day. The
treatment regi~e ~ay require administration over extended
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periods of time. The amount per administered dose or the
total amount ~Am; n; .ctered will be determined by the
physician and depend on such ~actors as the nature and
severity o~ the disease, the age and general health of the
patient and the tolerance o~ the patient to the co~pound.
The principles, pre~erred embodiments and modes of
operation o~ the present invention have been described in
the ~oregoing speci~ication. The invention which is
intended to be protected herein, howe~er, is not to be
construed as li~ited to the particular forms disclosed,
since they are to be regarded as illustrative ~ather than
restrictive. Variations and changes ~ay be made by those
skilled in the art without departing from the spirit o~ the
invention.
CA 02224867 1997-12-li
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~yu~-r~u-~ LISTING
F~~r lN~-okhATIoN:
ti) APPrICANT: Ha~e, John T~
M~Yell2r, r' ~n C.
(ii1 TITrE OF l~v~ ON: O~esity Protein InteL ~ ~ i ~te~ and Th~ir
Pr~par~ion and U~e
( iii ) N~ K OF ~U~S: 19
( iV ) ~ l ~~KI~.' yU~ Ar ~.~ .- c.-~
~A) ~n~PFCCF~ Eli Lilly and ~
~B) ~ Lilly CO~ ~Le Center
rTy: Tn~i ~n~polis
~D) STATE: Tn~i~n~
~E) ~G~h-~: United States
tF) zrP: 46Z8S
~v) ~. ru.~K ~F~n~RT T-' FORM
'A) MEDIU~ TYPE: Floppy di~k
'B) ~OMPUTER: ~BM PC r-ompatible
C) OPT~TTNG Sra-~: PC-DOS~MS- W S
'D) SOPTWARE: Pat~ntIn R~1Q~e ~1.0, Version #1.30
tYi ) r -~ APP ICATTON DATA:
(A) APPE~2 ~~ hu~.~K:
~B) FILING DATE:
~ C ) rr.- c cTFIcA~Io~
(~Lii) ATTORNEY/AGENT lN ~-oh~-ATIoN:
~A) NANE: Caltrider, ~ P.
~B) REGISTRATION NU~ 36 ~ 467
~C) K~ ~: X--lOO59
( iX ) 'I r:~ ~ro~uN~r~ToN INFORMATION:
~A) 5~3LEPHO~E: 317-276-0757
~B~ TELEFA;X: 3L7--2~7--1917
(2~ LN~ .A~rTO~ FOR SEQ rD NO:l:
~Q~JENCE C~RACTF~rSTICS
) ~ENC5~: 146 ~mino ~CidS
!B) r~ : aninO aCid
C) ~TP~N~ P:~ Sing~e
, D ) TOPOLOGY: 1 j ~ ~r
( ii ) ~Ot-F~-F~ : PrOt~in
CA 02224867 1997-12-17
WO97/00~86 PCT~S96/10613
-43-
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 28
(D) OTHER INFORMATION: /note= "Xaa at position 28 is Gln
or absent:"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
Leu Thr Ser Met Pro Ser Arq Asn Val Ile Gln Ile Ser Asn ~.sp Leu
Glu Asn Leu Arq Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
l00 105 llO
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140
Gly Cys
145
(2) INFORMA~ION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 amino acids
(B) TYPE: amino acid
(C) S~RANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME~KEY: Modi~ied-site
(B) LOCATrON: 28
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W097/00886 PCT~US96/10613
-44-
(D) OTHER IN~O~TION: ~note= "Xaa at position 28 is Gln
or absent:"
~xi) S~yu~:~CE DESCRIPTION: SEQ ID NO:2:
VaL Pro Ile Gln Lys Val Gln Asp Asp T~r Lys ~r Leu Ile Lys Thr
l 5 l0 15
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xa~a ~er Val Ser Ser
Lys Gln Lys Val T~r Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
~ 35 40 45
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
Leu T~r Ser Met Pro Ser Arq Asn Val Ile GLn Ile Ser Asn Asp Leu
65 70 75 80
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
go 95
(2) lN~-OR~ATION FOR SEQ ID NQ:3:
( i ) S~!;UU~ ; rlTAl~Ar~rF~2r~ cs
'A) LENGTX: 49 amin~ acids
B) TYP~: amino acid
,'C) STRAND~-N~ single
D ) TOPOLOGY: 1 i n~r
(ii ) Ir-TF~lr ~ TYPE: peptide
(xi) S~u~:N~ DESCBIPTION: SEQ ID NO:3:
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
l 5 l0 l~
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
~eu Gln Gly Ser Leu GLn Asp Me~ Leu Trp GLn Leu Asp Leu Ser Pro
Gly
(~) INFO~MA~T~N FOR 5~Q ID NO:4:
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-45-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Gly Ser His Met
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 a~ino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Gly Ser Ser His His Hi6 His His His Ser Ser Gly ~u Val Pro
l 5 l0 15
Arg Gly Ser His Met
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) S~RANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Leu Gl~ Lys Arg Glu Ala Glu Ala
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-46-
l 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: ~mino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Glu Ala Glu Ala
(2) INFORNATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACT~RISTrCS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) SIRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Leu Glu Lys Arg
~2) INFORMATION FOR SEQ ID NO:g:
(i) SEQUENCE CHARACTERIS~ICS:
(A) LENG~: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: sin~le
(D) TOPOLOGY: linear
( ii ) M~T.~CUr ~ TYPE: protein
(xi) SEQUENCE DESCRIPTrON: SEQ ID NO:9:
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Met Gly Ser Ser His His His His ~is His Ser Ser Gly Leu Val Pro
l 5 10 15
Ar~ Gly Ser Pro
(2) INFORMATlON FO~ SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(C) STRAND~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Gly Ser Pro
(2) INFORMATION FOR SEQ ID NO~
(i) SEQUENCE CHARACTERISTICS:
'A) LENGTH: 146 amino acids
B) TYPE: amino acid
C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATlON: 28
(D) OTHER INFORMATION: /n~te= "Xaa at position 28 is Gln
or absent'l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys ~hr Leu Ile Lys Thr
l 5 l0 15
lle Val Thr Ar~ Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
Lys Gln Lys Val ~hr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
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W097/00886 PCT~S96/10613
-48-
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
go 95
His Leu Pro Gln Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly
l00 105 ll0
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125
Leu Gln Gly Ser Leu Gln Asp Met Leu Gln Gln Leu Asp Leu Ser Pro
130 135 140
Gly Cys
145
t2) INFORMA~ION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l46 amino acids
(B) TYPE: a~ino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Val Pro Ile Trp Arg Val Gln Asp Asp T~r Lys Thr Leu Ile Lys Thr
l 5 l0 15
Ile Val Thr Ar~ Ile Ser Asp Ile Ser His Met Gln Ser Val Ser Ser
Lys Gln Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Val
Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Ala Ile Tyr Gln Gln Ile
Leu Thr Ser Leu Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys
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Pro Leu Pro Gln Ala Arq Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly
l00 105 ll0
Val Leu GLu Ala Ser Leu Tyr Ser Thr Glu Val Val ~la Leu Ser Arg
115 120 125
Leu Gln Gly Ala Leu Gln Asp Met Leu Arq Gln Leu Asp Leu Ser Pro
130 135 140
Gly Cys
145
(2) INFORMA~ION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: L46 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modi~ie~-site
( B ) LOCATrON: 28
(D) OTHER rNFoRMATIoN: /note= ~'Xaa at position 28 is Gln
or absent."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:L3:
Val Pro Ile Cys Lys Val Gln Asp Asp Thr Lys ~hr Leu Ile Lys Thr
l 5 l0 15
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
Lys Gln Ar~ Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Leu
Leu Ser Leu Ser Lys Met Asp Gln ~hr Leu Ala Ile Tyr Gln Gln Ile
Leu Thr Ser Leu Pro Ser Ar~ Asn Val Val Gln Ile Ser Asn Asp Leu
Glu Asn Leu Ar~ Asp Leu Leu His Leu Leu ALa Al~ Ser Lys Ser Cys
Pr~ Leu Pro Gln Val Ar~ Ala Leu Glu Ser Leu Glu Ser Leu Gly Val
l00 105 ll0
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Val Leu Glu Ala Ser Leu Tyr Ser Thr G1U Val VaL Ala Leu Ser Arq
115 120 125
Leu Gln GlV Ser Leu Gln Asp Met Leu Arq Gln Leu Asp Leu Ser Pro
130 135 140
Gly Cys
145
(2) INFORMATION FOR SEQ ID NO:l4:
;uu~NcE CHARACTERlSTICS~
(A) LENGTH: 146 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: sin~le
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 140
(D) OTHER INFORMATION: /note= "Xaa at position l40 is Gln
or absent."
(xi) ~Uu~CE DESCRIP~ION: SEQ ID NO:14:
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
l 5 lO 15
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
Glu Asn Leu Ar~ Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
9o 95
Val Leu Glu Ala Ser Gly ~yr Ser Thr Glu Val Val Ala Leu Ser Arg
lOO 105 ll0
Leu Gln Gly Ser Leu Gln Asp ~et Leu Trp Gln Leu Asp Leu Ser Pro
115 L20 125
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Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser
130 135 140
Gly Cy6
145
t2) INFORMATION FOR SEQ ID N~:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l46 amino acids
(B) TYPE: amino acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( ii ) M~T F~Ur ~ TYPF: protein
(xi) ~u~NcE DEscRIpTroN: SEQ ID NO:15:
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
l 5 lO 15
Ile VaL Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
Lys G~n Lys Val ~hr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
Leu Thr Leu Ser Lys Met Asp Gln ~hr Leu Ala Val Tyr Gln Gln Ile
Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
~5 so 95
His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
L00 105 ll0
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
LL5 120 125
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 135 140
Gly Cys
145
(2) INFORMATION FOR SEQ ID NO:16:
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NCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids
(B) ~YPE: a~ino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) ~uu~cE DEscRrpTIoN: SEQ ID NO:16:
Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
1 5 10 15
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Al~ Val Tyr Gln Gln Ile
Leu Thr Ser Met Pro Ser Arq Asn Val Ile Gln Ile Ser Asn Asp Leu
Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys
His Leu Pro Gln ~'a Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly
lOO 105 llO
Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro
130 13S 140
Gly Cys
145
(2) INFORMATION FOR SEQ ID NO:17:
(i) S~u~ CHARACTERISTICS:
(A) LENGTH: 146 amino acids
(L) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
.
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Val Pro lle Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr
1 5 lO 15
Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser
Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro GLy Leu His Pro Ile
Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
Leu Thr Ser Met Pro Ser Ar~ Asn Val Ile Gln Ile S~r Asn Asp Leu
G1u Asn Leu Arq Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser cys
His Leu Pr~ Gln Ala Ser Gly Leu GlU Thr Leu Asp Ser Leu Gly G.i~
100 105 llO
Val Leu GlU Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
115 120 125
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp GLn Leu Asp Leu Ser Pro
130 135 140
G~ Cys
~45
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: singLe
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GGGGCATATG AGGGTACCTA TCCAGAAAGT CCAGGATGAC A 41
(2) rNFoRMATIoN FOR SEQ ID NO:l9:
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(i) ~u~CE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SLQ ID NO:l9:
GGGGGGATCC TATTAGCACC CGGGAGACAG GTCCAGCTGC CACAACAT 48
