Fig. 3

Optimization of induction medium for improving recombinant protein yield from P_PEPCK. A Visualization of RBD secreted into medium by KpP_PEPCKRBD^* cultured in BMY-MSG and BMEY for 24 h and 48 h by SDS‒PAGE. His-tagged RBD from 5 ml culture medium was bound to Ni-agarose beads and analyzed by SDS‒PAGE. The pH of the culture medium after 24 h and 48 h is indicated. H₂SO₄ was added to BMY-MSG after 24 h of induction to maintain pH < 7.5. M, molecular weight markers (kDa). B Analysis of the growth of KpP_PEPCKRBD^* cultured in BMY-MSG and BMEY. Data are the average of two independent experiments. C Extracellular pH measured at different time intervals when KpP_PEPCKRBD^* was cultured in BMY-MSG, BMEY and BMEY-MSG. D Visualization of RBD secreted into 5 ml of medium by KpP_PEPCKRBD^* cultured in BMEY and BMEY-MSG for 24 h, 48 h and 72 h by SDS‒PAGE. His-tagged RBD bound to Ni-agarose beads was analyzed by SDS‒PAGE. M, molecular weight markers (kDa). E Analysis of the growth of KpP_PEPCKRBD^* cultured in BMEY, BMEY-MSG and BMY-MSG. Data are the average of two independent experiments. F Visualization of RBD secreted into 10 ml of medium by KpP_PEPCKRBD^* cultured in BMEY-MSG (INDI-1) and BMEYU-MSG (INDI-2) for 24 h, 48 h and 72 h by SDS‒PAGE. His-tagged RBD bound to Ni-agarose beads was analyzed by SDS‒PAGE. M, molecular weight markers (kDa). The growth of cells measured by absorbance at A₆₀₀ as well as the pH of the culture medium are indicated. M, molecular weight markers (kDa). G Comparison of RBD yield from KpP_AOX1RBD, KpP_PEPCKRBD and KpP_PEPCKRBD^* cultured for 72 h in BMMY or INDI-2 as indicated. One milliliter of culture medium was incubated with Ni-agarose beads, and RBD bound to the beads was eluted, estimated using Bradford reagent and examined by SDS‒PAGE. M, molecular weight markers (kDa). H Quantification of data presented in G. RBD was estimated using Bradford reagent from a standard curve generated from a known concentration of bovine serum albumin. Data are the average from three independent experiments (n = 3). Error bars in the graphs denote the mean ± S.D. Culture media are shown in parentheses. I Analysis of the growth of KpP_AOX1RBD, KpP_PEPCKRBD and KpP_PEPCKRBD^*. Culture media are shown in parentheses. Data are the average from three independent experiments (n = 3). J Comparison of GFP yield from KpP_AOX1GFP, KpP_PEPCKGFP and KpP_PEPCKGFP^* cultured for 72 h in BMMY or INDI-2 as indicated. GFP was purified from whole cell lysates of 0.2 ml cells using glutathione-S-transferase (GST)-tagged anti-GFP nanobodies. GFP bound to GST-tagged anti-GFP nanobodies was visualized by SDS‒PAGE. M, molecular weight markers (kDa). K Quantitation of GFP bands in the gel by densitometric scanning using ImageJ. Data are the average from three independent experiments (n = 3). Error bars in the graphs denote the mean ± S.D. The numbers in parentheses indicate the volumes of culture used for purification of the recombinant protein (RBD/GFP). In all experiments, the carbon source was replenished every 24 h