The production of recombinant 63kDa glycoprotein of Leishmania major by methylotropic yeast Pichia Pastoris

authors:

avatar AliAkbar Shabani , * , avatar Robert MakMaster , avatar Bahram Kazemi , avatar Mohsen Karimi , avatar Delavar ShahbazZade , avatar Ferydon Mahbodi

Koomesh: Vol.3, issue 1; 69-82
published online: February 15, 2002
article type: Research Article

How To Cite Shabani A, MakMaster R, Kazemi B, Karimi M, ShahbazZade D, et al. The production of recombinant 63kDa glycoprotein of Leishmania major by methylotropic yeast Pichia Pastoris. koomesh. 2002;3(1):e151946. 

Abstract

Introduction: Cut aneo us lesihmahiasis (L) is a world wide infect io us diseas e . Several approach toward vaccine have been taken around the world . Recombinant vaccines using gp63 in cocta il form is one of the candidates. Because, a significan t protection against a challeng with L. major was elicited in senssitive BALB/C mice after vaccination with recombinant BCG producing gp63, as well as , gp63 delivered orally by salmonella typhimurium C an preferably induce th e development of Th-I subset of CD4+ T cells. Since expression of eukaryotic genes are similar to Pichia pastoris as a eukaryotic cell, refolding and glycosylation may be an alogous to native form. GP63 gene from L. major (NIH strain) cloned and expressed in Pichia pastoris . Materials and Methods: Modified gp 63 ge ne th at encoded mature protein o nly (478 aa ) cloned in shuttle vector pHIL-Sl. Under contro l of alcohol oxidase 1 gene promotor (pAOXI) with yeast acid phosphatase signal sequence (PHO I). KM71 and GSI 15 strains of Pichia pastoris transformed with it and selected on histidin e minus medium. PCR and Southerbn blotting were done on chromosomal DNA of transform ant s yeast. Expressio n of rgp63 was evaluated by using Northern blotting, SDS-PAGE, Western-blott ing and immuno electro n micro sco py. Its activity was evaluated by using SDS-PAGE gelatin gel. Results: PCR and Southern blott ing a na lys is o n chromosomal DNA o f t rnsfo rma nts have bee n demons tra ted th at gp63 gene in te grated into chro mosomal DNA of ye as t. Expressio n of rgp63 in transformants of Pichia past oris confirmed by using Nor thern blott ing and SDS-PAGE. The findings were cinfirmed by Western blotting analysis and immunoelectron microsco py too. Conclusion: Rgp63 expressed in Pichia pastori s had glcosylated form too. It was act ive on SDS-PAGE gelatin gel. Thus, it was more similar to native gp63.