Fig. 5

Production and purification of the human growth hormone (hGH) using a TIR^EVOLVED. a Production levels of hGH using five different signal peptides. In each instance the difference between TIR^UNEVOLVED/TIR^EVOLVED pairs was assessed by immuno-blotting. The TIR^EVOLVED had originally been selected for β-lactamase (see Fig. 2). In this experiment, protein production was induced for 2 h, then a volume of cells corresponding to 0.2 OD₆₀₀ units of cells were harvested, separated by a 12% SDS-PAGE and protein levels were determined by immuno-blotting with antisera to a poly-histidine tag. ‘Pre’ denotes the precursor form of the protein and ‘Mat’ denotes the mature version. b An overview of the methodology used to purify hGH. c Analysis of the purified hGH by Size-Exclusion Chromatography (SEC). d Purified hGH was analysed by SDS-PAGE under denaturing- and non-denaturing conditions. e Activity of the purified hGH by using the MTS cell proliferation assay