Stability improvement of human collagen α1(I) chain using insulin as a fusion partner

Abstract

To enhance the stability of recombinant human collagen α1(I) chains (rhCOL1A1) in production and purification stages, a gene fragment fusing COL1A1 and insulin protein coding domains was synthesized and inserted into the pPIC9K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation. After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1(I) chain fusion protein (INS-COL1A1) reached about 300 mg·L− 1, and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni2 +-chelating chromatography. A prominent improvement in the stability of INS-COL1A1 was observed compared to rhCOL1A1 in vitro, and the rhCOL1A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins.