Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-06-04
2001-02-27
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S070100, C435S071100, C435S254200, C435S254230, C435S255100, C435S255500, C435S252300, C435S069800, C435S471000, C435S320100, C435S483000, C435S325000, C435S360000, C435S366000
Reexamination Certificate
active
06194169
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods of protein production, purification and characterization. The invention more particularly relates to the expression of platelet-derived growth factor (PDGF), especially in the yeast
Pichia pastoris.
BACKGROUND OF THE INVENTION
Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant that plays critical roles in embryogenesis, cell differentiation and wound repair. PDGF is a family of three disulfide-linked glycoprotein dimers of M
r
29,000-32,000 that arise from stochastic assembly of the homologous subunits, A-chain and B-chain, yielding the heterodimer PDGF-AB and homodimers PDGF-AA and PDGF-BB [for a review, see ref. 1]. The A-chain is expressed as two different forms (“long” and “short”) as determined by alternative splicing, with the long form containing an extended carboxyl terminus of basic amino acids that mediates association with the extracellular matrix and is encoded by exon 6 [2]. The B-chain also contains a cationic carboxyl terminus that has been implicated in the strong association of PDGF-BB with the cell surface [3]. The activity of PDGF is mediated by two PDGF receptor isotypes, &agr; and &bgr;, which upon ligand binding associate noncovalently to form homo- and heterodimers [4,5]. The PDGF &agr;-receptor has a high affinity for both A- and B-chains, while the &bgr;-receptor recognizes only the B-chain [6].
X-ray crystallographic analysis of recombinant human PDGF-BB has revealed an antiparallel arrangement of the disulfide-linked B-chain monomers [7]. Each B-chain monomer is characterized by a so-called “cysteine knot” structure, consisting of three intrachain disulfide bonds and two antiparallel &bgr;-strands, interconnected by three loop segments (I-III). The cationic residues Arg
159
, Lys
160
, Lys
161
of the A-chain, which correspond to the loop III region of the B-chain, are demonstrated as being required for high affinity receptor binding and mitogenic activity [8]. Furthermore, a synthetic peptide containing loop III residues 157-163 of the B-chain exhibits PDGF antagonizing activity [9], while a mutation at residue Lys
161
of the B-chain is shown to interfere with high affinity binding to the &agr;-receptor [10].
The present invention concerns the transformation of
Pichia pastoris
with a cDNA encoding the PDGF B chain. Others have used the
Pichia pastoris
system to express proteins. For instance, U.S. Pat. No. 5,324,639 discloses the expression of insulin-like growth factor-1 in
Pichia pastoris
cells. The recombinant protein is disclosed as being secreted into the culture medium at the level up to 100 times higher than results published in the literature using
Saccharomyces cerevisae
as the host cell. However, the reference does not suggest expressing PDGF in this system.
Cook et al., “Purification and Analysis of Proteinase-Resistant Mutants of Recombinant Platelet-Derived Growth Factor-BB Exhibiting Improved Biological Activity,”
Biochem. J
. 281: 57-65 (1992) disclose the expression of recombinant platelet-derived growth factor-BB and two protease-resistant mutants thereof in
Saccharomyces cerevisiae
. The expression construct utilized sequences encoding the yeast &agr;-factor pre-pro sequence upstream of the PDGF coding sequences to facilitate secretion. However, secretion into the culture medium was very low and most of the PDGF remained intracellular. The authors state that “a major limiting factor to PDGF-BB expression is, therefore, the secretion of PDGF-BB into the medium.”
Craig et al., “Characterization of the Structure and Conformation of Platelet-Derived Growth Factor-BB (PDGF-BB) and Proteinase-Resistant Mutants of PDGF-BB Expressed in
Saccharomyces cerevisiae,” Biochem. J
. 281: 67-72 (1992) disclose structural studies of the recombinant PDGF-BB produced in Cook et al. (above). The authors conclude that the protease-resistant mutants have the same secondary and tertiary structures as the wild-type PDGF-BB. The structure is reported to be considerably different than that reported for authentic PDGF-BB.
Duan et al., “A Functional Soluble Extracellular Region of the Platelet-Derived Growth Factor (PDGF) &bgr;-Receptor Antagonizes PDGF-Stimulated Responses,”
J. Biol. Chem
. 266: 413-418 (1991) disclose expression of the PDGFR extracellular domain in Chinese Hamster Ovary cells using an amplifiable marker to increase expression. The publication discloses that the protein was expressed in the culture medium at a level of 12 mg/L. The extracellular domain was extensively glycosylated, and bound to PDGF-BB with an affinity similar to that of the intact PDGF receptor. However, the reference does not teach or suggest expression of a PDGF protein in
Pichia pastoris.
There remains a need to increase the efficiency of producing proteins generally, and PDGF proteins in particular. It is expected that with improved production methods the cost of these proteins can be brought down and their availability for treating a wide range of conditions can be increased. PDGF proteins are particularly implicated in as being useful in such applications as cancer therapy, wound healing, and the treatment of gastrointestinal ulcers.
SUMMARY OF THE INVENTION
The present invention is directed to a method of significantly increasing the efficiency of producing platelet-derived growth factor (PDGF) proteins, and thereby reducing their cost. The method is exemplified with human PDGF-BB, both wild-type and mutant forms. According to the principles of the present invention, wild or mutant PDGF is produced by transforming
Pichia pastoris
with a vector encoding the mature protein, maintaining the yeast under predetermined culture conditions, and isolating the protein from the supernatant. The PDGF proteins produced by this method are indistinguishable from corresponding mature PDGF proteins produced in humans or other systems.
A preferred aspect of the invention is where the PDGF protein assembles as a PDGF BB homodimer. More particularly, the PDGF BB protein is mammalian, preferably human, in origin. The PDGF protein can be wild-type or a mutant thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present invention permits enhanced yields and production efficiencies of platelet-derived growth factor (PDGF) proteins. The PDGF can be in either a wild or mutant form. In particular, the invention entails producing the PDGF by transforming
Pichia pastoris
with a vector encoding the mature protein. Preferably, the vector encoding the mature protein is an expression vector, which further encodes a secretory peptide signal fused to the PDGF protein in order to facilitate secretion of the protein from the cells. The transformed cells are maintained under predetermined culture conditions for a desired length of time, and the protein is then isolated from the supematant. Whenever a signal peptide is employed, it is preferred that the signal is removed, e.g., proteolytically, either in vivo or in vitro prior to isolation of the mature protein so that a cleavage step can be avoided. As discussed herein, the PDGF proteins produced by this method are indistinguishable from corresponding mature PDGF proteins produced in humans or other systems.
In a preferred embodiment of the invention, the PDGF protein assembles as a PDGF BB homodimer. Preferably, the PDGF BB protein is mammalian, more preferably human, in origin. The PDGF protein can be wild-type or a mutant thereof. Contemplated mutants of PDGF include single amino acid substitutions, insertions and/or deletions, as well as multiple mutations. Exemplary of multiple mutations are two, three and higher substitutions of the residues occurring naturally in the wild protein.
Culture conditions effective to express PDGF in the transformed yeast are exemplified by those set forth hereinafter. A representative temperature range for maintaining the culture is 25-35° C., preferably 30° C. A representative time for conducting the culturing is 2-4 days, pref
Hamud Fozia
McDermott & Will & Emery
Mertz Prema
The University of Kentucky Research Foundation
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