Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1995-06-07
2001-11-27
Saoud, Christine J. (Department: 1647)
Drug, bio-affecting and body treating compositions
Lymphokine
C514S008100, C530S351000, C930S145000
Reexamination Certificate
active
06322779
ABSTRACT:
TECHNICAL FIELD
The invention relates to processes for purification and refolding of bacterially produced recombinant proteins in forms having high specific biological activity. In particular, it concerns procedures which make possible the production of biologically active, dimeric forms of CSF-1 from bacterial hosts expressing genes encoding the monomer.
BACKGROUND ART
Colony stimulating factor-1 (CSF-1) is one of several proteins which are capable of stimulating colony formation by bone marrow cells plated in semisolid culture medium. CSF-1 is distinguished from other colony stimulating factors by virtue of its ability to stimulate these cells to become predominantly macrophage colonies. Other CSFs stimulate the production of colonies which consist of neutrophilic granulocytes and macrophages; predominantly neutrophilic granulocytes; or neutrophilic and eosinophilic granulocytes and macrophages. A review of these CSFs has been published by Dexter, T. M.,
Nature
(1984) 309:746, and by Vadas, M. A.,
J Immunol
(1983) 130:793. There is currently no routine in vivo assay which is known to be specific for CSF-1 activity.
The characteristics of native human CSF-1 are complex, and in fact it is not yet clear what form of CSF-1 is active in the human body. Soluble forms of naturally-produced CSF-1 have been purified to various degrees from human urine, mouse L-cells, cultured human pancreatic carcinoma (MIA PaCa) cells, and also from various human and mouse lung cell conditioned media, from human T-lymphoblast cells, and from human placental-conditioned medium. Many, if not all of the isolated native CSF-1 proteins appear to be glycosylated dimers, regardless of source. There is considerable variety in the molecular weights exhibited by the monomeric components of CSF-1, apparently the result of variations in C-terminal processing and/or the extent of glycosylation. For example, Western analysis shows that the CSF-1 secreted by the MIA PaCa cell line contains reduced monomers of approximately 26 and 30 kd, as well as 40, 48, and 70 kd forms. Other CSF-1 molecular weights have been reported. For example, the monomeric reduced form of CSF-1 isolated from human urine is reported to be of the relatively low molecular weight of 25 kd when isolated, and 14-17 kd when extensively deglycosylated in vitro (Das, S. and Stanley, E. R.,
J Biol Chem
(1982) 257:13679).
The existence of “native-like” CSF-1 reference proteins is important because these proteins provide standards against which to compare the quality and biological activity of refolded recombinant forms of CSF-1. For this purpose, we have relied upon the soluble CSF-1 produced by the Mia PaCa cell line as well as properties of other highly purified CSF-1 molecules which have been described in the literature. The specific activity of these purified “native-like” reference proteins has typically fallen in the range of 4 to 10×10
7
units per mg (as measured by in vitro mouse bone marrow colony-forming assays).
CSF-1 has also been produced from recombinant DNA using two apparently related cDNA clones: (1) a “short” form which encodes a message which, when translated, produces a monomeric protein of 224 amino acids preceded by a 32-amino acid signal sequence (Kawasaki, E. S., et al,
Science
(1985) 230:292-296, and PCT WO86/04607, both of which are incorporated herein by reference); and (2) a “long” form, encoding a monomeric protein of 522 amino acids, also preceded by the 32-amino acid signal sequence. The long form has been cloned and expressed by two groups, as disclosed in Ladner, M. B., et al,
The EMBO J
(1987) 6(9):2693-2698, and Wong, G., et al,
Science
(1987) 235:1504-1509, both of which are incorporated herein by reference. (The DNA and amino acid sequences for both “short” and “long” forms are shown in
FIGS. 5 and 6
, respectively; however, the 32 amino acid signal sequence is incomplete as illustrated in
FIG. 6.
)
The long and short forms of the CSF-1-encoding DNA appear to arise from a variable splice junction at the upstream portion of exon 6 of the genomic CSF-1-encoding DNA. When CSF-1 is expressed in certain eucaryotic cells from either the long or short cDNA forms, it appears to be variably processed at the C-terminus and/or variably glycosylated. Consequently, CSF-1 proteins of varying molecular weights are found when the reduced monomeric form is analyzed by Western analysis.
The amino acid sequences of the long and short forms, as predicted from the DNA sequence of the isolated clones and by their relationship to the genomic sequence, are identical with respect to the first 149 amino acids at the N-terminus of the mature protein, and diverge thereafter by virtue of the inclusion in the longer clone of an additional 894 bp insert encoding 298 additional amino acids following glutamine 149. Both the shorter and longer forms of the gene allow expression of proteins with sequences containing identical regions at the C-terminus, as well as at the N-terminus. Biologically active CSF-1 has been recovered when cDNA encoding through the first 150 or 158 amino acids of the short form, or through the first 221 amino acids of the longer form, is expressed in eucaryotic cells.
Since most, if not all, of the native secreted CSF-1 molecules are glycosylated and dimeric, significant posttranslational processing apparently occurs in vivo. Given the complexity of the native CSF-1 molecule, it has been considered expedient to express the CSF-1 gene in cells derived from higher organisms. It seemed unlikely that active protein would be obtained when the gene was expressed in more convenient bacterial hosts, such as
E. coli
. Bacterial hosts do not have the capacity to glycosylate proteins, nor are their intracellular conditions conducive to the refolding, disulfide bond formation, and disulfide-stabilized dimerization which is apparently essential for full CSF-1 activity. Thus, experimental production of recombinant CSF-1 in
E. coli
has, prior to this invention, resulted in protein of very low activity, although its identification as monomeric CSF-1 had been readily confirmed by immunoassay, N-terminal sequencing, and amino acid analysis.
It is by now accepted that inactive forms of recombinant foreign proteins produced in bacteria may require further “refolding” steps in order to render them useful for the purposes for which they are intended. As a dimeric protein containing a large number of cysteines and disulfide bonds, which are required for activity, CSF-1 represents a particularly difficult challenge for production from bacterial systems. Often, recombinant proteins produced in
E. coli
, including CSF-1 so produced, are in the form of highly insoluble intracellular protein precipitates referred to as inclusion bodies or refractile bodies. These inclusions can readily be separated from the soluble bacterial proteins, but then must be solubilized under conditions which result in essentially complete denaturation of the protein. Even secreted proteins from bacterial sources, while not necessarily presenting the same solubility problems, may require considerable manipulation in order to restore activity. Each different protein may require a different refolding protocol in order to achieve full biological activity.
A number of papers have appeared which report refolding attempts for individual proteins produced in bacterial hosts, or which are otherwise in denatured or non-native form. A representative sample follows.
Reformation of an oligomeric enzyme after denaturation by sodium dodecyl sulfate (SDS) was reported by Weber, K., et al,
J Biol Chem
(1971) 246:4504-4509. This procedure was considered to solve a problem created by the binding of proteins to SDS, and the process employed removal of the denatured protein from SDS in the presence of 6 M urea, along with anion exchange to remove the SDS, followed by dilution from urea, all in the presence of reducing agents. The proteins which were at least partially refolded included: aspartate transcarbamylase, B-galactosidase, rabbit muscle aldolase, and coat pro
Cowgill Cynthia
Halenbeck Robert
Koths Kirston
Laird Walter J.
Blackburn Robert P.
Chiron Corporation
Morley Kimberlin L.
Pochopien Donald J.
Saoud Christine J.
LandOfFree
Recombinant human CSF-1 dimer and compositions thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant human CSF-1 dimer and compositions thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant human CSF-1 dimer and compositions thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2569825