The LARGE-SCALE PROTEIN PRODUCTION Team was headed by Brian Fox, PhD, and was responsible for large-scale protein production and labeling for the project.
The major goal of the Protein Production Team was to reliably produce sufficient mass of bacterial cells expressing recombinant proteins to facilitate subsequent protein purification efforts. By working closely with the Small-Scale Expression, Protein Purification, and X-ray Crystallography teams, deliverable goals of 15 g of recombinant cell paste with favorable scoring for total expression, solubility of expressed protein, and percentage of proeolysis from the fusion construct using tobacco etch virus protease were established.
The Team produced SeMet-labeled proteins by heterologous expression in Escherichia coli grown using the auto-induction approach introduced by Studier [Protein production by auto induction in high density shaking cultures and Recipes and stock solutions described in protein production by auto induction in high density shaking cultures]. As compared to other methods for growth and induction of recombinant cultures, we found the auto-induction method to be reliable and scalable for all types of expression monitoring used at CESG. Moreover, for large-scale cell growth, the auto-induction minimizes the amount of labor required for culture monitoring. As described below, our implementation of the large-scale cell growth pipeline was sufficiently robust that all proteins studied by CESG are labeled with selenomethionine (SeMet) as the first-pass production effort.
Pipeline Production of SeMet-Labeled Proteins in Auto-Induction Medium. We developed a cell-based protein production pipeline that incorporates the auto-induction strategy introduced by Studier for first-pass production of SeMet-labeled proteins in Escherichia coli B834. The entire production cycle from receipt of freshly transformed expression host through the growth, induction, and expression was designed to take ~72 h, and capacity for up to 36 growths per week has been demonstrated with existing labor and equipment. The medium used for culture scale-up was adjusted to provide rapid and reproducible growth, while the production cell growth was optimized for timing, cell mass yield, total protein expression, and percentage of SeMet incorporation. At 250 rpm shaking in a standard refrigerated shaker, an average final optical density at 600 nm of greater than 6 and a yield of ~14 g of cell paste were obtained over ~500 growths. Cell growth with shaking at 350 rpm gave an ~1.6 fold increase in OD600 and a corresponding increase in the mass of cell paste with similar characteristics for the expressed fusion proteins (total expression, solubility, and proteolysis of the fusion protein to release the target). Increased agitation indicates a potential pathway to improvements in process yield. However, it was not possible to fully load the shaker at this higher agitation rate due to overheating before the 24 h growth cycle could be completed. Engineering efforts to improve the shaker and rack design were researched.
Scoring of Protein Expression.CESG uses three-tier scoring to evaluate expression gels: (1) total protein expression, (2) the level of soluble expression, and (3) the percentage of total soluble fusion protein that can be proteolyzed by tobacco etch virus (TEV) protease. This scoring is based on visual comparison with the stained intensity in standard lanes. Similar assessments are made for the lanes containing the soluble fraction, the pellet fraction, and the TEV protease-treated soluble fraction. MALDI-TOF mass spectrometry was used to identify endogenous E. coli proteins useful as control markers for cell fractionation. By including evaluation of these internal control proteins to assure consistency in cell lysis, the CESG scoring system was successfully standardized across the expression of several hundreds of targets.