Secreted Expression Of The Human Recombinate Interleukin 2 In Pichia Pastoris And Its Large-scale Fermentation And Purification

Interleukin-2(IL-2)is the first pharmaceutical grade protein bygenetic engineering that can gain high profit in China.In 1976,Morganfound in the supernatant fluid of spleen cells there was a cytokinewhich can promote and maintain T cell growth in vitro,the cytokinewas called T cell growth factor,then was named Interleukin-2 in 1979.In 1980,Rosenberg discovered that IL-2 can induce LAK proliferation,then people combinated IL-2 and LAK to treat tumor patient in advancedstage and gained certain therapeutic effect.Taniguchi succeeded incloning cDNA of IL-2 in 1983 from Jurkat cell after stimulated byConA.Then people are deeply interested in the biological functionand clinical application of IL-2.Recently,people know more aboutarchitecture and function of IL-2,meanwhile,discover gradually manynew clinical application.IL-2 is composed of 4 pieces of exons and 3 pieces of introns.The gene of IL-2 locate in 4q26-27,about 5kb.Molecular weight is15kDa,pI is 6.6～8.2.The maturate IL-2 is composed of 133 amino acidsand has no homology with other cytokine.Regulation of its geneticexpression is in the genetic transcriptional level.IL-2 is a globularprotein and composed of 4 main amphoteric α spirals,the latter alignantiparallelly,the hydrophobic surface constructs a hydrophobiccore.The biologic activity of IL-2 is regulated by the recepotor inthe cellular membrane.The receptor is made up of three glycoproteinsubunits,that is:α,β and γ chain.Molecular weight is separately55kDa,75kDa and 64kDa.So the subunits are separately called p55,p75and p64.The chain of αcombine with IL-2 with low affinity and haveno signal transmit function.IL-2Rγ dosen't combine with IL-2 alone,when IL-2Rγ chain bind with IL-2Rα,β chain or bind with IL-2Rβchain, IL-2 has high affinity.Signal transmission is concerned withβ and γ chain,more related with γ chain.IL-2 is a kind of lymphocyte factor,can promote cytotoxicity Tcell,NK cell and lymphokineactivated killer cell multiplication andreinforce their activity.Meanwhile IL-2 can promote lymphocyte tosecret antibody and interferon.IL-2 have many functions,such as:antivirus,antitumor and enhancing immunologic function of organism.At present,in clinic IL-2 is used of treating malignant tumor,suchas,renal cell carcinoma,melanoma,hepatoma,pulmonary carcinoma andso on;controlling breast-abdominal dropsy,treating inherent oracquired immunity and autoimmunity,such as rheumatoid arthritis,systemic lupus erythematosus.Furthermore,IL-2 can treat some kindof virus,bacillus and cystoendophyte infectious diseases,such as,lepriasis,pulmonary tuberculosis,blastomyces albicans infectiousdiseases.IL-2 has very important applicable value because of its extensivebiological effect.The common method is to construct recombinantgenetic engineering bacteria of IL-2.By mass culture and annealing,purification of expression product,we can obtain IL-2 with highspecific activity.Now,E. coli expression system is the most commonmeans and the technique is better-provided.But this technique hasa lot of disadvantages,isn't fit for commercial production in largescale.For this reason,we plan to use Pichia pastoris to expressrecombinant IL-2,to try to establish an effective and stabileexpression system for its clinical research.1. Synthesis of rhIL-2 geneWe change the genetic codons of mature peptide to Pichia pastorisfavorite codons by artificial synthesis and insert XhoⅠtargetsite,Kex2 in 5'end,insert terminal codon taa and EcoRⅠtarger sitein 3'end. Kex2 is essential to cut α signal peptide efficiently.Cloning vector is pBLuescript Ⅱ SK,cloning site is XhoⅠ/EcoRⅠ,host bacterium is XL1-Blue.Inoculate some host bacterium in LB medium containing Amp, shakein 37℃ overnight, then extract synthetic recombinant plasmidPBS-rhIL-2 by Plasmid extraction Kit in accordance with directions.2. Screening and appreciation of Pichia pastoris strain thatsecreted express rhIL-2 stably and effectively.1)Construction of eukaryotic expression vector pPICZα2-rhIL-2Recombinant plasmid PBS-rhIL-2 is cut by XhoⅠ/EcoRⅠ,afterpurification inserted in the pPICZα2 vector which is cut by the sameenzymes.After confirmation by endonuclease digestation assay andsequencing, recombinant vector is linearizated by BstXⅠ,thentransform it into the X-33 via electrotransformate.2)Screening of recombinant Pichia pastoris strainAfter electrotransformation yeast fungus was spread on YPDliquid medium containing Zeocin,culturing in 30℃ for about 72h.Thenwe extract the genomic DNA of the transformed yeasts viaboiling-freezing-boiling method to perform PCR using the AOX1 5'and AOX1 3'primers.3)Expression and assay of rhIL-2Proliferate the PCR tested positively yeast clones,then inducedthe expression of rhIL-2 with methanol.Perform SDS-PAGE to identifyrhIL-2.The results indicated that there was a 15kDa protein in thesupernatant.3. Studies on large-scale fermentation and purification process ofrhIL-2:1)Studies on large-scale fermentation process of rhIL-2:Pichiapastoris has many advantages as a kind of expression host,and it'svery suitable for large-scale expression of the extraneous proteins.We explored the large-scale fermentation process of rhIL-2 and foundthat the best pH is pH5.0,DO between 25%～30% and the supply speedof methanol is 9 ～ 10ml/h/L initial fermentation volume.Theconcentration of rhIL-2 in the broth can reached 50mg/L.2)A new method to purify rhIL-2 at large-scale: The rhIL-2 couldbe purified by SP Sepharose XL on pH4.0.Then by SourceTM30RPC.Theyield coefficient was about 55%.So, our studies succeed in secreted expression of IL-2 in Pichiapastoris.Established initially expression system of IL-2.ViaSDS-PAGE we discovered there was a 15kDa protein in the supernatant.It's about 50mg/L.To applying,we should consummate fermentationcondition.