Chen D.,Beijing A BZYMO Biosciences Co. |
Li L.,Beijing A BZYMO Biosciences Co. |
Ban J.-Y.,Beijing A BZYMO Biosciences Co. |
Guo Y.-Z.,Beijing A BZYMO Biosciences Co. |
And 4 more authors.
Chinese Journal of Biologicals | Year: 2017
Objective: To achieve high-level secretory expression of recombinant human pepsinogen I (PG I) in Hansenula polymorpha and prepare the purified recombinant PG I into a calibrator for in vitro diagnostic reagents. Methods: According to the H. polymorpha-prefered codon, PG I gene was designed, synthesized and cloned into expression vector pRMHP2. 1. The constructed recombinant plasmid was transformed to H. polymorpha 26012 by electroporation for several rounds of passage and stabilization, and the strain in which PG I was highly expressed in a secretory form was screened and identified for the integrated site and copy number of heterologous gene. Fermentation liquid was prepared in a 200 L fermenter, from which recombinant PG I protein was purified by nickel ion affinity chromatography and determined by a latex-enhanced immunoturbidimetric kit. The purified PG I protein was prepared into calibrators at concentrations of 100 and 50 μg/L respectively and tested for stability. Results: Restriction analysis and DNA sequencing proved that recombinant plasmid pRMHP2. 1-PG I was constructed correctly. After rounds of screening, a recombinant H. polymorpha strain was obtained, in which PG I protein with a relative molecular mass of about 45 000 was expressed. The yield of expressed product was more than 100 mg/L, while PG I gene of was integrated into the host rDNA site, and the gene copy number was not less than 20. The purity of purified recombinant PG I protein was 94. 5%. The mean degradation limit of active ingredient contents of PG I as a calibrator in accelerated stability, real time stability and on-board stability tests were not more than 10%. Conclusion: Recombinant PG I protein was highly expressed in a secretory from in H. polymorpha, which showed high stability and might be used as a calibrator for in vitro diagnostic reagents.
Liu J.,Beijing Abzymo Biosciences Co. |
Chen D.,Beijing Abzymo Biosciences Co. |
Li L.,Beijing Abzymo Biosciences Co. |
Liu X.-Y.,Beijing Abzymo Biosciences Co. |
And 6 more authors.
Chinese Journal of Biologicals | Year: 2012
Objective: To synthesize Macaca mulatta granulocyte-macrophage colony stimulating factor (mGM-CSF) gene, highly express in E. coli and purify the expressed product. Methods: According to the E. coli-preferred codon, mGM-CSF gene was designed and synthesized, and cloned into prokaryotic expression vector pET-43.1a (+). The constructed recombinant plasmid pET-43.1a-mGM-CSF was transformed to E. coli BL21-CodonPlus (DE3)-RIPL and induced with IPTG. The expressed recombinant mGM-CSF was purified by Sephacryl S-200 molecular sieve chromatography, re-naturalized, then determined for reactogenicity by Western blot, and for biological activity by MTT method. Results: Both restriction analysis and sequencing proved that recombinant plasmid pET-43.1a-mGM-CSF was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 15 000, contained about 30% of total somatic protein and mainly existed in a form of inclusion body. The protein reached a purity of more than 95% after purification and re-naturalization, and showed specific binding to rat anti-human GM-CSF monoclonal antibody. The specific activity of the recombinant protein was 1.2 × 107 IU/mg. Conclusion: Recombinant mGM-CSF was successfully expressed in E. coli, which showed high biological activity after purification and re-naturalization.