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. 2017 Oct 17;199(22):e00342-17. doi: 10.1128/JB.00342-17

FIG 4.

FIG 4

Purification, molecular mass, and flavin cofactor analysis of recombinant Fe-S d-iLDH from P. putida KT2440. (A) SDS-PAGE analysis of the purified recombinant Fe-S d-iLDH. Lane M, molecular mass markers; lane 1, crude extract of E. coli C43(DE3) carrying the empty pETDuet-1 plasmid; lane 2, crude extract of E. coli C43(DE3) carrying the pET-lldE plasmid; lane 3, sample obtained from Ni2+-nitrilotriacetic acid (NTA) chromatography; lane 4, sample obtained from DEAE anion-exchange chromatography; lane 5, sample obtained from gel filtration chromatography. (B) Gel filtration chromatography of Fe-S d-iLDH on a Superdex 200 10/300 GL column. The standard molecular mass markers are thyroglobulin (660 kDa), ferritin (440 kDa), catalase (250 kDa), aldolase (158 kDa), lactic acid dehydrogenase (140 kDa), bovine serum albumin (67 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa). Ve/V0, ratio of elution volume to void volume of the column. (C) HPLC analysis of the flavin cofactor of Fe-S d-iLDH. (D) The fluorescence of authentic FAD before and after treatment with phosphodiesterase I. (E) The fluorescence of authentic FMN before and after treatment with phosphodiesterase I. (F) The fluorescence of flavin cofactor of Fe-S d-iLDH before and after treatment with phosphodiesterase I.