Extended Data Fig. 2: PAGE analysis of tobacco Rubisco production in E. coli BL21-Star.
From: Laboratory evolution of Rubisco solubility and catalytic switches to enhance plant productivity
PAGE analysis of a single experiment examining the soluble protein from a tobacco (Nt) leaf and E. coli BL21-star expressing wildtype (WT) and mutant tobacco L8S8 Rubiscos (coded by plasmid P2, Fig. 1c) and tobacco chloroplast chaperones (plasmid P3) were separated by native-PAGE and (a) stained with Coomassie or (b) blotted onto nitrocellulose and immuno-probed with tobacco Rubisco antibody. In addition to the A242V substitution promoting L8S8 holoenzyme production, it facilitated the production of a larger RbcL intermediary complex (*), previously shown to contain RAF1 (R1) and/or BSD2 (B2) subunits (L8(R1/B2)n28. (c) Corresponding SDS PAGE separation of the same soluble (S) protein fractions and corresponding total (T) cellular protein (comprising soluble and insoluble membrane and aggregated/inclusion body proteins) showing the Cpn60 and Cpn20 produced are mostly fully soluble while only a small proportion ( < 20%) of the RbcL, RbcS and Rca produced in E. coli are soluble (+), more so in those containing the A242V mutation in RbcL (++). Asmbl refers to the expression of only P3 plasmid; EV, empty pCDF plasmid; GroE, E. coli GroELS chaperonin cages; CPN, tobacco Cpn60α/β/20 chaperonin complexes; CPN + L, CPN encaged RbcL (slightly visible in panel b). Uncropped images of the original gels and blot are provided in Source Data Fig. 2 and Extended Data Figs. 2, 4, 6 and 7.
