The model equations of the production phase of rHSA fermentation were derived on the base of both elemental balance and metabolic balance, then the unknown parameters of the model were estimated by multivariable optimization. The possible reasons of discrepancy of production rate between different period of fermentation were discussed. The model could preferably described the relations between different macroscopic reaction rates of the process and keys for the high-efficiency expression of HAS were deduced.
Fermentation ; physiology ; Humans ; Models, Biological ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

In order to obtain enough fusion protein for developing preclinical studies of IFNbeta-HAS, we screened Pichia pastoris transformants expressing high-level protein by immunology method. The yield of IFNbeta-HSA was about 500 mg/L by fed-batch fermentation. The purity of IFNbeta-HSA reached 96% through the steps of ultrafiltration, Blue Sepharose FF, Ni2+-IMAC and DEAE Sepharose FF. Analysis of Western blotting showed that IFNbeta-HSA had the antigenicity of IFNbeta and HSA. The specific activity was about 1.96 x 10(7) IU/mg by standard survival activity test on WISH cells challenged with VSV virus. This study provided a method to produce IFNbeta-HSA.
Fermentation ; Humans ; Interferon-beta ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serum Albumin ; biosynthesis ; genetics

Human serum albumin (HSA) is an important biological product in clinical pharmacy practice at present. Its main clinical use is in maintaining colloid osmotic pressure and increasing circulating plasma volume so as to cure hemorrhagic shock, burn, cancer, hypercytosis, hypoalbuminosis, etc. HSA is isolated by fractionating human plasma, which entails possible contamination by viruses or prions. Recombinant human serum albumin (rHSA) has been successfully produced by biological engineering. The structural and functional properties of rHSA are similar to those of plasma derived human serum albumin (pdHSA). Preclinical and clinical trials have confirmed the safety and efficacy of using this rHSA preparation in the treatment of certain diseases.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Serum Albumin ; biosynthesis ; genetics

OBJECTIVETo obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris.

METHODSHSA and PTH(1-34) cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GS115 competent cells treated with LiCl, and mut+ transformants were screened on MD plate. With AOX promoter and alpha-MF signal sequences leading, fusion protein was expressed in GS115. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA-PTH(1-34). The fusion protein was identified by Western blotting and classical adenylate cyclase assay.

RESULTThe PCR results showed that the gene of HSA-PTH(1-34) was integrated into GS115 genome. Western bolt approved the existence of two domains of HSA and PTH(1-34). The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH (1-34) activated adenylate cyclase, but the activity was lower than that of the synthetic PTH(1-34).

CONCLUSIONActive fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.

Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Peptide Fragments ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics ; Teriparatide ; analogs & derivatives

In order to make a large-scale preparation of(GLP-1A2G)2-HAS (GGH), the double-plamid pPICZalphaB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR to create the recombinant expression plasmid pPICZalphaB-ggh, which was transformed into P. pastoris GS 115/F2 that was integrated by another recombinant expression plasmid pPIC9K-ggh. The immunology method combined with high concentration antibiotic was used to screen recombinant strain P. pastoris GS115/F3 capable of high-level expression of GGH protein. The GGH fusion protein expressed by GS115/F3 increased 49.7% compared with the GS 115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 degrees C. At the same time, the quantitative PCR results showed that GGH gene dose in GS115/F3 increased 26.7% with respect to that of GS 115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.
Genetic Vectors ; genetics ; Glucagon-Like Peptide 1 ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction ; methods ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

To increase the in vivo half-life of human CNTF mutein AX15 (R13K), HSA-AX15 (R13K) fusion protein was constructed by the fusion of the C-terminus of HSA to the N-terminus of AX15 (R13K) via an 11 amino acids linker. HSA-AX15 (R13K) fusion protein was purified to homogeneity by cation exchange chromatography, reverse phase chromatography and gel filtration after expressed in pichia pastoris. TF-1 cell survival bioassay showed the biological activity of AX15 (R13K) was not affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8 mg/kg HSA-AX15 (R13K) fusion protein could produce more potent anti-obesity effects on KM mice than daily injection of 1.6 mg/kg AX15 (R13K). The long-acting form of hCNTF variant has the potential to reduce discomfort by requiring fewer injections and to minimize the side-effects by decreasing the dosage and fluctuation of plasma concentration, and thus has superior clinical application.
Animals ; Ciliary Neurotrophic Factor ; genetics ; Humans ; Mice ; Mutant Proteins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serum Albumin ; genetics

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.
Chimera ; Gene Deletion ; Gene Knockout Techniques ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Mannosyltransferases ; genetics ; Pichia ; enzymology ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics

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.
Animals ; Bioreactors ; microbiology ; Fermentation ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; Macaca fascicularis ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacokinetics ; Recombinant Proteins ; Serum Albumin ; biosynthesis ; genetics

Adult ; Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Culture Media ; Female ; Hepatitis, Viral, Human ; blood ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Serum ; Serum Albumin ; biosynthesis ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo investigate the causes and influencing factors of heterogeneity of HSA/IL1ra fusion protein expression in Pichia pastoris.

METHODSThe heterogeneity of HSA/IL1ra fusion protein expressed in Pichia pastoris was studied by removing glycosylation and inhibiting glycosylation, as well as by different ways of fusion, different clones, and different expression host.

RESULTGlycosylation caused expression heterogeneity of fusion protein, but in SMD1168 and some GS115 clones there was no this phenomenon.

CONCLUSIONThe expression heterogeneity of HSA/IL1ra fusion protein in Pichia pastoris is due to the glycosylation, and different ways of fusion, different clones, different expression host also have some impact.

Escherichia coli ; genetics ; metabolism ; Genetic Heterogeneity ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics