Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Patent
1996-04-25
1999-06-15
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
530399, 530416, 530417, C07K 710, C07K 114, C07K 320
Patent
active
059123290
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method for purification of a mixture of hydroxamate derivatized protein and native protein by treating the mixture with immobilized metal and thereby adsorb the hydroxamate derivatized protein on the immobilized metal and recover the native protein. The protein is e.g. IGF-I.
INTRODUCTION
One main problem when proteins are produced in expression system is related to the folding of the protein to its native configuration. Many expression systems lead to the production of aggregates of denatured proteins, so called inclusion bodies, of which only a part will lead to the desired native protein.
General methods to facilitate and render the refolding effective has been found. One is the use of a class of heat-shock-proteins (HSP) and the other is folding-enzymes. By using HSP, aggregation is avoided and by using the folding enzymes, the speed of refolding is accelerated.
Another suggested method for the recovering of the native protein is solubilization of the inclusion body protein with a denaturant, such as guanidine or urea and if needed a reduction of the disulphide bond. By dilution or dialysis and reoxidation, the protein can be refolded to the native protein.
U.S. Pat. No. 5,151,501 (American Cyanamid) discloses a process for solubilization and naturation of somatropins (Growth hormones) by dispersing somatropin refractile bodies in a solution containing sulfolane and thereafter dilution.
Human recombinant IGF-I has been produced as a secreted product in both Escherichia coli and Saccharomyces cerevisiae. In isolated material from both species, IGF-I is found mainly as mis-folded forms with intermolecular disulfide bonds. In addition, in vitro refolding of reduced IGF-I in the presence of oxygen, has demonstrated that native, mis-matched and aggregated IGF-I accumulate, even under dilute refolding conditions.
A promising system is expression of a hybrid-IGF-I molecule in a prokaryotic cell (Escherichia coli) as host organism. This system creates however other problems. Mammalian polypeptides expressed in prokaryotic cells occasionally form aggregates (inclusion bodies). Such aggregates or inclusion bodies are associated with improper formation of intermolecular bonds. The general approach that has been used for obtaining biologically active polypeptides is to treat the inclusion bodies with chaotropic agents and reducing agents--to refold the polypeptides into their native form. Successful refolding, without formation of new inclusion bodies, is generally difficult at high concentrations of the recombinant protein. The best yield is generally achieved at concentrations around 20-200 .mu.g/mL. Refolding is therefore considered to be a very expensive production form that demands a cost intensive drug.
With Escherichia coli expressing the hybrid protein Z-IGF-I, very high expression levels have been achieved (6-7 g/l fermentation). Reference is here given to EP 230 869, especially the examples. The present bottleneck for this system is to remove the Z part in order to achieve native IGF-I after refolding.
When the recombinant construction Z-IGF-I is connected by the peptide bond of asparagine-glycine, the Z part can be efficiently removed to 70-80%, by treating the heterologous IGF-I with high concentrations of hydroxylamine (1-2M, pH 9.5) (Nilsson B, et al. Methods in Enzymology, vol 198, 1991). Unfortunately does IGF-I contain the amino acids Gln15, Asn 26 and Gln 40 that also have the potential to chemically react with hydroxylamine and form hydroxamate (Bornstein P and Balian G. Methods in Enzymology, 47, 132-145, 1977). That this unwanted side reaction actually occurs has been confirmed by Canova-Davis et al. ( Biochem J, 285, 207-213, 1992).
Even gentle treatment seems to produce too much of this side product. It is therefore creating a purification and separation requirement.
Theoretically seven forms of IGF-I can be formed during hydroxylamine cleavage with three different molecular weights ;.DELTA.16, .DELTA.32 and .DELTA.48. Canova-Davis et al., 1992 reported three fo
REFERENCES:
Ramadan, N., Porath, J. Iron(3+)-hydroxamate as immobilized metal affinity-adsorbent for protein chromatography. J. Chromatogr., 321(1), 93-104, Jan. 1995.
Porath, J. IMAC-immobilized metal ion affinity based chromatography. Trends in analytical chemistry, 7, 254-259, Jul. 1988.
Farkas et al Complexes of peptide hydroxamates. Complex formation between transition metals and L-proplyl-L-leucylglycinehydroxamic acid and L-prolyl-L-leucinehy, Mar. 1990.
Blackburn Robert P.
Borin Michael
Chiron Corporation
Tsang Cecilia J.
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