Figure 5

Expression in E.coli. (A) eGFP was SLIC cloned into different E.coli pCoofy vectors using standard LP1 (3C) and LP2 (ccdB) primers and expressed in BL21 Rosetta (DE3) at 24°C. 2ml cultures were harvested and lyzed in PBS + protease inhibitors at consistent cell density / buffer ratio. eGFP expression level in total lysate was monitored on Agilent BioAnalyzer P80 Chips; eGFP fluorescence in cleared lysate was recorded on a LightCyclerII instrument. (B) Expression results for different target proteins cloned in pCoofy1 (His₆), pCoofy2 (His₆Trx), pCoofy3 (His₆GST) and pCoofy4 (His₆MBP) plus pCoofy35 (MBP only). Proteins are classified as soluble, insoluble or not expressed. “Soluble” proteins could be enriched by IMAC and were further purified with or without tag. Proteins classified as “insoluble” were not soluble in standard IMAC buffers (buffer A). These constructs were either subject to refolding, buffer screens or discontinued. Proteins classified as “not expressed” cover constructs, that were expressed at low or even undetectable level in Western Blot analysis and were thus discontinued. (C) Pil1 (UniProt: P53252). SAQ and SFK mutants were SLIC cloned into pCoofy1 (His₆), pCoofy2 (His₆Trx), pCoofy3 (His₆GST) and pCoofy4 (His₆MBP) using standard LP1 (3C) and LP2 (ccdB) primers and expressed in BL21 Rosetta (DE3) at 30°C. oD3 samples (0,1 ml culture of oD₆₀₀ of 3 is lyzed in 50μl sample buffer) were loaded on Agilent BioAnalyzer P80 Chips. Expression levels indicated are based on relative peak quantification.