High-level Expression Of Long Chain Arg~3-IGF1 In Pichia Pastoris And Its Purification And Characterization

Human Insulin like Growth Factor 1(huIGF1) are single-chain polypeptides with structural homology to proinsulin.They regulate proliferation and differentiation of a multitude of cell types.Unlike insulin,they are produced by most tissues of the body and are abundant in the circulation.The absence of huIGF1 will cause much diabetes,so as it plays an important role in treating diabetes.A number of recombinant fusion protein analogs of IGF-â… ,in which the N-terminal region has been modified,show increased biological activity.Reduced binding to the IGFBPs rather than enhanced binding to the IGF receptors is the basis for the increased activity.One of these analogs,Long-[Arg3]-IGF-I(LR~3IGF-1),has Glu~3 in native IGF-â… replaced by Arg~3,while the N terminus has a 13-amino acid extension consisting of the first 11 amino acids of methionyl porcine growth hormone followed by the dipeptide Val-Asn.The secondary structure of the IGF-â… domain of Long-[Arg3]IGF-â… was to be almost identical to the native protein,so it is presumed that the bioactivity of LR~3IGF-1 will be almost identical to the native huIGF1.The use of the methylotrophic yeast,Pichia pastoris,as a cellular host for the expression of recombinant proteins has become increasing popular in recent times.P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities.Equally important,P.pastoris is also a eukaryote,and thereby provides the potential for producing soluble,correctly folded recombinant proteins that have undergone all the post-translational modifications required for functionality.Additionally,linearized foreign DNA can be inserted in high efficiency via homologous recombination procedures to generate stable cell lines whilst expression vectors can be readily prepared that allow multiple copies of the target protein, multimeric proteins with different subunit structures,or alternatively the target protein and its cognate binding partners,to be expressed.A further benefit of the P.pastoris system is that strong promoters are available to drive the expression of a foreign gene(s) of interest,thus enabling production of large amounts of the target protein(s) with relative technical ease and at a lower cost than most other eukaryotic systems.In our research the cDNA coding for the long chain Arg~3-IGF-1(LR~3IGF-1) was synthesized and cloned into the secreting expression vector pPICZaA,and transformed the Pichia pastoris KM71 strain.The positive clones were selected,and after induction, an apparent molecular weight of 8kDa protein was found in the supernatant.The recombinant protein was purified to greater than 95%using the cation exchange chromatography and then Superdex 75 size-exclusion chromatography steps.Finally, 20mg of the protein was obtained in high purity from 500ml supernatant.Bioaetivity of the recombinant LR~3IGF-1 was confirmed by the ability to stimulate NIH 3T3 proliferation in vitro.As there were three disulfide bonds in the molecule of IGF1,so the preciseness partnership of these bonds were important to the protein structural parameters and characterized for biological activity.Our results suggest that the P.pastoris expression system can be used to produce LR~3IGF-1 for both research and industrial purpose. Finally,40mg of the protein was obtained in high purity from 1L supernatant.The recombinant protein was purified to greater than 95%using the cation exchangechromatography and then Superdex 75 size-exclusion chromatography steps. Bioactivity of the recombinant LR~3IGF-1 was confirmed by the ability to stimulate NIH 3T3 proliferation in vitro.