The gene hNGFB encoding the β subunit of human nerve growth factor (hNGF) was cloned into P. pastoris secretive expression vector pHIL-S1 and E. coli expression vector pET-15b. The recombinant hNGFB vectors pSNGF and...The gene hNGFB encoding the β subunit of human nerve growth factor (hNGF) was cloned into P. pastoris secretive expression vector pHIL-S1 and E. coli expression vector pET-15b. The recombinant hNGFB vectors pSNGF and pET15b-NGF were transformed into P. pastoris host cell GS115 (Mut^+ , His^- ) and E. coli strain BL21 (DE3) respectively. Expression and secretion of hNGFB in P.pastoris was attempted under the direction of the AOX1 promoter and PHO1 signal sequence. The positive colonies growing on medium without histidine were further selected by PCR. The yield of rehNGFB in GS115 was about 14.4% of total cellular secretive protein. The secreted protein was immunological active on Western blotting with rabbit anti-mNGFB antibodies. The fusion protein yield of rehNGFB in E. coli BL21 (DE3) was about 10.3% of total cellular protein after IPTG induction. Western blot detection showed its immunological activity.展开更多
To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted int...To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted into the pPIC9 K 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-COL1 A1) 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 Ni^(2+)-chelating chromatography. A prominent improvement in the stability of INS-COL1 A1 was observed compared to rhCOL1 A1 in vitro, and the rhCOL1 A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1 A1 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.展开更多
文摘The gene hNGFB encoding the β subunit of human nerve growth factor (hNGF) was cloned into P. pastoris secretive expression vector pHIL-S1 and E. coli expression vector pET-15b. The recombinant hNGFB vectors pSNGF and pET15b-NGF were transformed into P. pastoris host cell GS115 (Mut^+ , His^- ) and E. coli strain BL21 (DE3) respectively. Expression and secretion of hNGFB in P.pastoris was attempted under the direction of the AOX1 promoter and PHO1 signal sequence. The positive colonies growing on medium without histidine were further selected by PCR. The yield of rehNGFB in GS115 was about 14.4% of total cellular secretive protein. The secreted protein was immunological active on Western blotting with rabbit anti-mNGFB antibodies. The fusion protein yield of rehNGFB in E. coli BL21 (DE3) was about 10.3% of total cellular protein after IPTG induction. Western blot detection showed its immunological activity.
基金Supported by the National Natural Science Foundation of China(21676214,21576160,21506171)Shaanxi Key Laboratory of Degradable Biomedical Materials Program(2014SZS07-K04,2014SZS07-P05,15JS105,15JS106,2014SZS07-Z01,2014SZS07-K03)Shaanxi R&D Center of Biomaterials and Fermentation Engineering Program(2015HBGC-04)
文摘To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted into the pPIC9 K 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-COL1 A1) 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 Ni^(2+)-chelating chromatography. A prominent improvement in the stability of INS-COL1 A1 was observed compared to rhCOL1 A1 in vitro, and the rhCOL1 A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1 A1 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.
