Purification And Characterization Of HSA-G-CSF Expressed In Pichia Pastoris

Human G-CSF is a secreted polypeptide of 174 amino acids having a molecular weight of approximately 18KD.It was isolated initially from a cancer cell line and its gene has been cloned,sequenced and expressed in different cell hosts by genetic engineering techniques.G-CSF possesses the capacity to stimulate the differentiation and proliferation of bone marrow stem cells to granulocytes.As such,it possesses the capacity to stimulate the body's protective capacities against infection by promoting the growth of polymorphonuclear neutrophils and their differentiation ending in maturity.It is thus capable of activating the body's prophylactic functions,and may be used in different pathological situations in which the number of neutrophils is abnormally low or in which the immune system needs to be strengthened.Such situations arise,for example,following cancer chemotherapy treatments,in transplantation,and especially bone marrow transplantation,or in leukopenic states.One of the drawbacks of currently available G-CSF lies in the fact that it is rapidly degraded by the body once administered.These phenomena of elimination and degradation in vivo hence constitute at present an obstacle to exploitation of the biological activity of the G-CSF as a pharmaceutical agent.A long-acting form of G-CSF,which has the potential to reduce discomfort by requiring fewer injections and possibly by minimizing the adverse events associated with peaks and toughs in plasma concentration that occur with daily injection.Human serum albumin(HSA)is the major protein component of human plasma and consists of a single nonglycosylated polypeptide chain of 585 amino acids with a molecular weight of 66.5 kDa.Due to its remarkably prolonging half-life,together with its wide in vivo distribution and its lack of enzymatic or immunological functions,HSA represents an optimal natural carrier for therapeutic peptides aimed at interacting with cellular or molecular components in vivo.The development of genetic engineering has opened up the possibility to obtain recombinant proteins without the danger of contamination by human pathogens,and at lower cost.HSA-G-CSF fusion protein was produced as a recombinant protein composed of recombinant human serum albumin genetically fused at its C-terminus to the N-terminus of G-CSF in Pichia pastoris.It will be a prospective molecular with greatly prolonged t1/2 in vivo,retaining bioactivity.The HSA-G-CSF fusion protein could be given less frequently than G-CSF to achieve similar therapeutic effects in patients.1.Construction of pPIC9-HSA-G-CSF recombinant expression plasmidHSA gene was amplified from cDNA library of human liver and the oligonucleotide sequence coding for a polypeptide linker -GGGGS- was introduced into the 3'- end of the sense-strain of the gene.G-CSF gene was also amplified from the same cDNA library.The two amplified products were firstly ligated with each other by T4 DNA tigase and then together ligated with pPIC9 expression vector.The recombinants were identified by endonuclease digestion(with Sal I,Xho I,BamH I and EcoR I)and the result indicated the target gene had been successfully cloned into pPIC9 vector.The pPIC9-HSA-G-CSF recombinant plasmid had been finally identified by sequencing.2.Transformation of Pichia and screening for Mut⁺ and Mut^s transformantsThe linearized pPIC9-HSA-G-CSF recombinant plasmids were digested by Sal I and transformed GS 115 competent cells by electroporation.Transformants growed on RDB plate without histidine after 48h -72h at 30℃.Picking up some transformants, cultivated them on MM and MD plates respectively.After 2-4 days of incubation at 30℃,the phenotype of these transformants were indentified and Mut⁺ transformants accounted for 90%in total transformants. 3.PCR identification of Pichia transformantThe PCR identification of the Pichia integrants was carried out using genomic DNA of Pichia recombinant clones as templates and 5'- AOXI and 3'- AOXI oligonucleotide fragments as primers.The PCR products were analyzed by DNA electrophoresis.Two bands respectively with size approximately 2.2kb,2.4kb could been seen.It demonstrated that the fusion gene of HSA-G-CSF had been integrated into Pichia genome.4.Expression of HSA-G-CSF recombinant proteinPicking up several identified recombinant colonies,cultivated in BMGY medium in shake flasks(250rpm/min)at 30℃.After one day of cultivation,the cells were transferred to BMMY medium and 100%methanol was added to final concentration of 3%for induction of the recombinant proteins every 24h.After additional 72h of cultivation,the cells were harvested and the supernatants were collected for expression analysis.SDS-PAGE results indicated the recombinant protein with 86kDa as expected had been expressed through secretion way and the production of recombinant protein increased with induced time extended.The yield of HSA-G-CSF at 72h was about 450mg/L.5.Purification of HSA-G-CSF fusion proteinThe fermentation supematant was purified through affinity column,hydrophobic column,ion exchange column and desalting column with AKTA Exerplorer.The purity of fusion protein exceeded 97%by HPLC analysis.6.Western blotting analysis of HSA-G-CSF fusion proteinWestern blotting result demonstrated that HSA-G-CSF fusion protein could be recognized by rabbit anti G-CSF polyclonal antibodies and anti-HSA antibodies.This result proved that the recombinant expression product was HSA-G-CSF fusion protein exactly.7.Structure identification of HSA-G-CSF fusion proteinThe structure of HSA-G-CSF fusion protein was identified by isoelectric focusing,amino acid sequencing,mass spectra and CD.The result indicated that the fusion protein simultaneously possessed amino acid sequences of HSA and G-CSF molecules.8.Bioactivity of HSA-G-CSF fusion proteinThe bioactivity of HSA-G-CSF fusion protein was determined by promoting the proliferation of NFS-60 cells.The results suggest that-the fusion protein could effectively stimulate the proliferation of NFS-60 cell with dose-dependent trend.9.Pharmacokinetics of HSA-G-CSF fusion proteinHSA-G-CSF fusion protein was administered subcutaneously(SC)to mice and the serum samples were collected at different times.The drug concentration in serum was analyzed by ELISA.Pharmacokinetic parameters were calculated with 3P87 software.The t_1/2,CL of the fusion protein was 10.6h,15ml/h/kg,respectively.A five-fold increase in plasma half-life and an eight-fold decrease in clearance were seen with HSA-G-CSF fusion protein compared to G-CSF protein alone.These results suggested HSA-G-CSF fusion protein had a better long-acting effect compared to G-CSF.Conclusions:Using western blotting,isoelectric focusing,amino acid sequencing,mass spectra and CD assay,HSA-G-CSF recombinant protein had been identified.The result of NFS-60 cell proliferation assay indicated that the fusion protein had a similar bioactivity with G-CSF.The pharmacokinetic studies of HSA-G-CSF fusion protein proved that the fusion protein had a better long-acting effect than G-CSF.These studies suggested that the fusion protein could be given less frequently than G-CSF to achieve similar therapeutic effects in patients.