Optimization of the hydroxylamine cleavage of an expressed fusion protein to produce recombinant human insulin-like growth factor (IGF)-I
✍ Scribed by Steven J. Milner; Sonia M. Thomas; F. John Ballard; Geoffrey L. Francis
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 868 KB
- Volume
- 50
- Category
- Article
- ISSN
- 0006-3592
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✦ Synopsis
The application of gene fusion technology for the production of heterologous proteins in Escherichia coli has required the development of specific cleavage methods to separate the coexpressed fusion protein partner from the protein of interest. When hydroxylamine is used to cleave Asn-Gly fusion protein linkages, undesirable chemical modification of asparagine and glutamine amino acids can also occur. In this study, hydroxylamine cleavage conditions were modified t o minimize unwanted chemical heterogeneity that occurred during the cleavage of the fusion protein [Met'l-pGH(1-11)-Val-Asn-IGF-l (Long-IGF-I). The cleavage reaction was shown t o be dependent on the hydroxylamine concentration, temperature, and pH. Optimal cleavage conditions were identified that resulted in very low levels of chemical heterogeneity, but under these mild conditions that cleavage of the labile Asn-Gly bond was reduced. Therefore, the reaction was further modified t o improve the yield of IGF-I while minimizing chemical heterogeneity. The yield of unmodified IGF-I was improved from less than 25% to greater than 70%. Analysis of the heterogeneity produced using the modified cleavage technique showed that Asn26 was converted to a hydroxamate. This variant was characterized in refolding and biological assays where it was equivalent to IGF-I. To further assess the effectiveness of the modified cleavage technique and to evaluate the potential for process scale-up, a gram-scale cleavage reaction of Long-IGF-I was carried out. The process yielded IGF-I with a low level of chemical heterogeneity that was easily removed by ion-exchange chromatography. Moreover, this work shows that the production of unmodified IGFs using hydroxylamine cleavage of fusion proteins is facilitated using the mild cleavage reaction.