Résumé
Yeast is a widely used host for recombinant protein expression. However, glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated. alpha-1,6-mannosyltransferases gene (och1) encodes the enzyme that initiates the first step of out-chain elongation of high mannose type N-glycan in yeast, which is different from that in human. So, a high efficient method to knockout target gene by two-step recombination was established and was used to delete och1. In the first recombinant, a plasmid with och1::ADE1 and ura3 gene was linearized in the downstream of och1 and inserted to the och1 site of P. pastoris genome, where the upstream and downstream of och1 were duplicated. In the second recombinant, the duplicated fragments of och1 were exchanged and the och1 deletion strains were selected on the plates containing 5-FOA, but no adenine. Then the och1 deletion strain was applied to express an human serum albumin (HSA) granulocyte-macrophage colony-stimulating factor (GM-CSF) chimera. Different with the hyperglycosylated HSA/GM-CSF chimera expressed in wild type P. pastoris, the chimera expressed in the och1 deletion strain, contained smaller N-glycan. The results suggested that the och1 mutant yeast may be more suitable for production of recombinant glycoproteins. And the och 1 deletion strain could be used for further re-engineering to produce complex human glycoproteins.
Sujets)
Chimère , Délétion de gène , Techniques de knock-out de gènes , Facteur de stimulation des colonies de granulocytes et de macrophages , Génétique , Mannosyltransferases , Génétique , Pichia , Génétique , Métabolisme , Protéines de fusion recombinantes , Génétique , Sérumalbumine , GénétiqueRésumé
To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.
Sujets)
Animaux , Humains , Bioréacteurs , Microbiologie , Fermentation , Interféron alpha , Génétique , Macaca fascicularis , Pichia , Génétique , Métabolisme , Protéines de fusion recombinantes , Génétique , Pharmacocinétique , Protéines recombinantes , Sérumalbumine , GénétiqueRésumé
Stem cell factor is an important hematopoietic growth factor. In this study, the human stem cell factor was produced by recombinant E. coli, and the structure and biological activity of the recombinant stem cell factor(rhSCF) was studied. It was indicated that the rhSCF was a uncovalent dimer in phosphate buffer,and had the correct mass spectra, mass peptides spectra, composition of amino acid, N-terminal sequernce, C-terminal sequence and intrachain disulfide linkages, rhSCF alone or synergy with rhG-CSF could mobilze hematopoietic progenitors to blood in monkey.