Fig. 2

Cloning, expression, purification and characterization of rmResistin. a: Construction of rmRetn expression plasmid, and arrow indicates the PCR product, of mResistin ORF, which contains 288 base pairs and codes 94 amino acids of mature mResistin with an additional initiating codon on 5’ terminus and a stop codon on 3’ terminus. M, marker; 1, PCR product of rmResistin expression plasmid verification. b: Compared with whole bacteria proteins without induction, a protein band, as arrow indicates, appears after induction with IPTG. M, marker; 1, whole bacterial proteins prior to induction; 2, whole bacterial proteins 4 h post induction with 1 mM IPTG. c: Inclusion bodies of rmResistin were dissolved in denaturing buffer of GdnHCl (lane 1) and renatured in renaturing buffer (lane 3). The impurities and unsuccessfully renatured inclusion bodies (lane 2) were removed. M, marker; 1, solubilized inclusion bodies; 2, pellet of centrifugation post renaturation; 3, the renatured protein solution. d: Loaded on the SP sepharose Fast Flow resins, rmResistin was washed and eluted with linear NaCl concentration from 0.02-1 M and the only UV280 peak, eluted out when conductivity reached, as the arrow indicates, shows rmResistin. Absorbance at 280 nm and conductivity were detected on monitor. The elution was fractioned into fraction 1, 2 and 3. e: Three fractions were subjected to 15 % SDS-PAGE and only one band with molecular weight about 10.4 KDa was observed in the three lanes; M, molecular weight marker; 1, fraction 1; 2, fraction 2; 3, fraction 3. f: Western blotting results of rmResistin. Only one band positioned at about 10.4 KDa was obverved and this was identified as rmRsisitin monomer. No dimmers, trimers or polymers of rmResistin were found. M, Molecular weight standard marker; 1, whole bacterial proteins containing induced rmresistin by IPTG; 2, purified rmResistin