Effect Of Two Different Secretion Signals On The Expression Of Canine Interferon In Pichia Pastoris

Interferon(IFN) has not only an antiviral activity, but also various kinds of biological activities including cancer therapy,immunomodulatory effects,cell growth inhibition,and differentiation-inducing activity. There are three types of interferons,type I IFN,type Ⅱ IFN and type Ⅲ IFN. Type I IFN(IFN-α) includes at least 20 different subtypes,it is used currently as a treatment against virus infections, and some metabolic or allergic diseases. Pichia pastoris has been used to produce recombinant Canine interferon α1(CaIFN-α1), the methanol-inducible AOX1 promoter drives the expression of heterologous protein,which is secreted intracellular or extracellular using the α-mating factor secretion signal. The Saccharomyces cerevisiae α-mating factor prepro peptide signal leader consists of pre-peptide and pro-peptide. The pre-peptide is believed to be important for interaction with the signal recognition particle,subsequent translocation into the endoplasmic reticulum(ER)and folding of the nascent protein,then itself be removed by signal peptidases. The pro peptide is thought to translocating the folding protein from the ER to the Golgi complex,and Kex2 endopeptidase severs the pro leader sequence, Ste13 protein rapidly cleaves the Glu-Ala(EA) repeats. The Saccharomyces cerevisiae α-mating factor prepro peptide is most commonly used signal sequence for CaIFN-α1expression in P. pastoris,however,a few studies have been done to determine the effect of mutagenesis on the activity of mutagenic α-mating factor prepro peptide.Here,we compared the secretion level of CaIFN-α1 in P. pastoris by α- factor signal and mutagenic α- factor signal to determine which is better for protein export.Canine interferon-alpha1(CaIFN-α1)was coloned and expressed in pPIC9 K under the control of alcohol oxidase promoter(AOX1) using Saccharomyces cerevisiae α-mating factor prepro sequence and this vector was named pPIC9K-CaIFN-α1. In this study,on the basis of pPIC9K-CaIFN-α1,a mutated αprepro sequence without the Glu-Ala(EAEA) repeats was designd for CaIFN-α1expression,and was named pPIC9K-muMFα-CaIFN-α1.pPIC9K-muMFα-CaIFN-α1 and pPIC9K-CaIFN-α1 were both lineared with salIrestriction enzyme. The lineared products were separately electrotransformed into Pichia pastoris GS115 using same performance,and two transformants we screened out by the same concentration of G418,then they were cultured in BMGY/BMMY medium. Total proteins in the supernant of cell culture were estimated by lowry protein assay and the Canine IFN-α1 was quantified by ELISA kit. The transformant with Saccharomyces cerevisiae α-mating factor prepro peptide signal was named rGS115 A,whereas with mutagenic α- factor signal was named rGS115 B. The total concentration of recombinant CaIFN-α1 in the culture supernatant of rGS115 A was found to be 131mg/mL,while in rGS115 B,it was 136mg/m L,there was no statistical significance between them(P>0.05),but expression quantity of Canine IFN-α1 was clearly different between rGS115 A and rGS115 B,the expression level of rGS115 B with mutagenic α- factor signal was 92mg/mL,took up 67.7% in total and rGS115 A with native secretion signal was 78mg/mL, took up 59.5%, expression level of rGS115 B was apparently higher than rGS115A(P<0.05).We further estimated biological activity of the two transformants on their antiviral activity and their immunologic activity using E-rossette forming test(checking T-lymphocytes percentage),and measured molecular weight of two bands by SDS-PAGE. The molecular weight of recombinant CaIFN-α1 secreted was 25 kDa,that was different from our prediction before(18.5kD). The reason we analyzed that CaIFN-α1 has two N-glycosylation sites,so it maybe modified by glycosylation in Pichia pastoris.The antiviral activity of CaIFN-α1 secreted by the two transformants was estimated,one was 2.58×107 u/mg(with Saccharomyces cerevisiae α-mating factor prepro peptide signal) and the other was 1.01×108 u/mg(with mutagenic α- factor signal) by VSV-MDCK inhibitory action assay. The activity of CaIFN-α1 with mutagenic α- factor signal was higher. Both of them had the effect on T cell proliferation using E-rossette forming test,but there was no statistical significance difference on T-lymphocytes percentage.In conclusion,in Pichia pastoris,the recombinant CaIFN-α1 with mutagenic α-factor signal is better than CaIFN-α1 with Saccharomyces cerevisiae α-mating factor prepro peptide signal on secretion efficiency and biological activity.