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Cloning and expression of CRES.

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posted on 19.02.2013, 19:46 by Li Wang, Qing Yuan, Sunhong Chen, Heng Cai, Meige Lu, Yue Liu, Chen Xu

(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.

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