Cloning and expression of CRES.

Abstract

<p>(A) Design of the oligonucleotide primers used to construct the cysteine mutants of CRES. (B) Schematic diagram of the recombinant plasmid PGEX-4T-1/CRES. (C) RT-PCR products of the CRES fragments on 1.5% agarose gel. Lane M, marker; lane N, negative control (PCR without cDNA); lane 1, CRES-N30 fragment; lane 2, CRES-N60 fragment; lane 3, CRES full length fragment. (D) RT-PCR products of the mutant CRES gene on 1.5% agarose gel. Lane M, marker; lane N, negative control; lane 1, CRES C105 mutant; lane 2, CRES C139 mutant; lane 3, CRES C105 & C139 mutant. (E) Prokaryotic expression and purification of CRES and GST recombinant protein. Lane M, marker; Lane 1, cells carrying the GST vector without the insert after Isopropylthio-β-D1-galctopyranoside (IPTG) induction; Lane 2–4, cells carrying the GST vector with the insert (CRES-N30,CRES-N60, full length CRES) after IPTG induction. Lane 5, purified GST recombinant protein. Lane 6–7, purified GST-CRES recombinant protein (N30, N60, N90). (F) Prokaryotic expression and purification of cysteine mutants of CRES. Lane M, marker; Lane 1, cells carrying the GST vector before IPTG induction; Lane 2–5, cells carrying the GST vector with the insert (wild type, C105 mutant, C139 mutant, C105 & C139 mutant) after IPTG induction. Lane 6–9, purified GST-CRES recombinant protein (wild type, C105 mutant, C139 mutant, C105 & C139 mutant). (G) The purified CRES recombinant proteins were analyzed by western blot using anti-GST monoclonal antibody.</p