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AMB Express
Fig. 2 | AMB Express

Fig. 2

From: NADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface

Fig. 2

Purification of LbADHWT and LbADHK71E. a Chromatogram of the affinity chromatography with the Strep-Trap™ HP column. b Chromatogram of the size-exclusion chromatography of the affinity-purified proteins with a Superdex™ 200 increase 10/300 GL column. c Calibration curve used for molecular mass determination obtained with proteins of known molecular mass: carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa) and β-amylase (200 kDa). For molecular mass determination, the partition coefficient Kav was calculated and plotted against the logarithm of the molecular mass. LbADHWT was shown to be tetrameric with a molecular mass of 107 kDa. LbADHWT eluted with an apparent molecular mass of 95 kDa and LbADHK71E with an apparent molecular mass of 113 kDa. d SDS-PAGE analysis of crude cell extracts (lanes 1 and 2) and soluble protein fractions (lanes 3 and 4) of E. coli C43(DE3)/pASK-IBA5plus-LbadhWT (lanes 1 and 3) and E. coli C43(DE3)/pASK-IBA5plus-LbadhK71E (lanes 2 and 4) and of purified LbAdhWT (lanes 5 and 7) and LbAdhK71E (lanes 6 and 8) after StrepTactin affinity chromatography (lanes 5 and 6) and after size-exclusion chromatography (lanes 7 and 8). The Mini-PROTEAN® TGX™ any kD™ gel was stained with GelCode™ Blue Stain Reagent (Thermo Scientific, Rockford, USA). M, protein molecular mass standards

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