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Cloning, Construction, Expression And Purification Of Human ~(17)Serine-Interferon-Beta Fusion Gene

Posted on:2005-08-23Degree:DoctorType:Dissertation
Country:ChinaCandidate:H S ChenFull Text:PDF
GTID:1100360122995854Subject:Cell biology
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Purpose:Human interferon-B is able to mediate antiviral, antiproliferative and immunomodulatory activities in response to a viral infection or other stimuli. Natural IFN-P is a 166-amino-acid glycoprotein, which has a single glycosylation site at residue Asn-80 and three cysteine residues. Cys-31 and Cys-141 form a disulfide bridge, which plays an important role in the stabilization of molecules. Cys-17 is free and can form an intermolecular disulfide bridge, which may reduce biological activity. It is also reported that the recombinant protein with serine-17 produced in E. coli is more stable with higher biological activities than that with Cys-17. The recombinant IFN-P therapeutic was approved for treatment of multiple sclerosis in 1993 by FDA .One of our aims is to modify human interferon-beta gene by substitution of the 17-Cysteine with serine. We also clon the original IFN-P gene in order to find their differenceThere are two recombinant IFN-B products in the past 20 years. One is produced in E.coli in insolvable form without glycosylation; the other is produced in Chinese hamster ovary (CHO) cells. The advantage of HuIFN-B produced in CHO cells is that the protein is identical with the natural one interms of primary structure and glycosylation pattern. However, the mammalian recombinant expression systems are of high cost and a low production. Now Pichia pastoris has been proved as an effective large-scale (fermentation) recombinant protein production tool. It can be easily manipulated at the molecular genetic level, express proteins at high levels intracellularly or extracellularly and perform many higher eukaryotic protein modifications, such as glycosylation, disulfide-bond formation, and proteolytic processing. The powerful genetic techniques make Pichia pastoris an economic system of choice available for heterologous protein expression. The other aim of our study is to express interferon-beta gene in Pichia pastoris and to find a new way for recombinant IFN-P products.There are some cases where STE13 cleavage of Glu-Ala repeats is not efficient, and Glu-Ala repeats are left on the N-terminus of the expressed protein of interest. In our study, Firstly, we insert genes rightly after Lys-Arg of a -factor mating signal sequence, which can avoid four residues remained. Secondly, We insert a His-tag and a cleavage site of Factor Xa between the secretion signal and IFN-P genes to express a His-tagged fusion protein. In this way, we can easily purify FuIFN-P by metal chelating affinity chromatography and the purified products can be further cleaved completely by the Factor Xa.Methods and results:1. Cloning and Construction of HuIFN-p, 17Ser-IFN-p and two fusionIFN-p genesIn our study, we amplify four IFN-P genes. The first one is original IFN-p gene (17Cys-IFN-p); The second one is I7Ser-IFN-p gene, We modified human interferon-beta original gene by substitution of the 17-Cysteine with serine; The third one is 17Ser-FuIFN-P, The fourth one is 17Cys-FuIFN-P,We insert a gene coding for a His tag and a Factor Xa cleavage site in front of IFN-P genes. These genes are cloned intoexpression vector pPIC9k and pPIC9K'. The recombinant plasmids are transformed into E. coli strain DH5a.Analysis of PCR, restrictive enzyme digestion and DNA sequence show that these genes have been cloned correctly and the recombinant plasmids are constructed successfully.2. Transformation and expression of four recombinant plasmids in Pichia pastorisRecombinant expression plasmids are digested by restrictive enzyme SacI, SalI and BglII, then transformed into Pichia pastoris GS115 cells by electroporation, LiCl and Pichia EasyComp?Kit. Patching His+ colonies on MD plates and the multicopy colonies on G418-YPD plates screen Pichia pastoris transformants. After isolation of total DNA from Pichia and PCR analysis of pichia intergrants, the strains are induced to express by methanol. The capacity to secrete rFuIFN-P of the selected clones was tested.We choose His+Mut+ transformants of GS115, linearized wit...
Keywords/Search Tags:Human-interferon-beta, Serine, Mutant, Fusion-gene, Pichia pastoris, Clon, Construction, Expression, Fermentation, Purification
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