Protein Expr Purif. present study showed that guanidine hydrochloride was more effective than urea in solubilizing the inclusion bodies. can also be a suitable host for production of antibody fragments with high concentrations up to 2 g/L (9). Due to these advantages, production of ScFv in can represent an effective bioprocess for large-scale manufacturing of antibody fragments (10). High-level expression of heterologous proteins in usually results in the formation of insoluble aggregates known as inclusion bodies (11). Inclusion bodies are resistant to proteolysis by proteases and can be easily harvested in a cost-effective downstream processing scheme (12). However, the yield of recombinant protein expression is dependent on a variety of biological and genetic parameters such as expression vector design, promoter strength, expression host strain, codon usage, as well as cultivation conditions (13). Optimization of cultivation conditions for overexpression of recombinant proteins are traditionally achieved by changing one variable at a time (14,15). This method is not only time-consuming, but also results in misinterpretation of the results when the conversation between different variables is present (16). There are many alternative approaches to simultaneously analyze several variables such as full factorial and fractional Gastrodenol factorial designs, response surface designs, and Taguchi method. Taguchi method and response surface methodology (RSM) are the most common multivariate analysis methods (17). Taguchi method can handle discrete variables however, it ignores parameter interactions (18). RSM can Gastrodenol be applied to determine the optimum culture conditions for protein expression by simultaneously changing several variables based on a minimum number of experiments. Furthermore Gastrodenol RSM can identify potential interactions among experimental variables, Mouse monoclonal to PRAK based on a reasonable prediction of culture conditions for optimal protein expression (19,20,21,22). Recently, we have developed Gastrodenol a new ScFv against HER2 (23). In the present study, we used RSM to optimize culture conditions for expression of this ScFv in by selecting three variables including isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration, temperature and post-induction time. In addition, the conversation between these variables on ScFv expression was investigated. The present work also evaluated alternative methodologies for ScFv harvesting and purification. MATERIALS AND METHODS Bacterial strains and plasmids The synthesized anti-Her2-ScFv gene was used as a template for polymerase chain reaction (PCR) to append BL21(DE3) pLysS (Novagen, Madison, WI, USA) using the CaCl2 method. A positive clone was sequenced to ensure no mutations and to confirm the correct reading frame for expression. Expression For expression of anti-Her2-ScFv protein, a positive recombinant clone was inoculated into 5 ml Luria-Bertani (LB) broth made up of 100 g/ml ampicillin and incubated at 37 C for 16 h at 180 rpm. This overnight culture was used to inoculate (1 % v?v) 50 ml of fresh LB medium in 250 ml Erlenmeyer flasks. Cells were incubated at 37 C until they reached the exponential phase (an OD600 nm of 0.4-0.6) then expression of protein was induced by addition of IPTG. Initial determination of the expression of anti-Her2-ScFv protein was performed with 1 mM IPTG at 37 C for 2 h. For the optimization of protein expression, each experiment was performed under various conditions of induction (IPTG concentration, post-induction time and heat) as described in experimental design. These variables have shown greatest effects on induction conditions in previous studies Gastrodenol (24,25). At the end of expression time, cell density of each sample (OD600) was measured and the cells were harvested by centrifugation at 7,500 g for 10 min. Experimental design and optimization of cultivation by response surface methodology To systematically evaluate the effects of three impartial variables including IPTG concentration (factor A), post-induction time (factor B) and heat (factor C), around the production of anti-Her2-ScFv in BL21(DE3) pLysS, an experimental design was developed using Box-Behnken factorial design scheme (26). Each variable was investigated at levels of -1 (the lower value of the variable), +1 (the higher value of the variable) and 0 (the central point of the variable) (Table 1). As a result, a total of 15 experiments were carried out including the one in triplicate at the center point (Table 2). Data analysis of experimental design and surface response methodology was performed using Design Expert software (version 8.0.7.1, Stat-Ease Inc., Minneapolis, USA). Table 1 Variables and levels used in the experimental design. Open in a separate window Table 2 BoxCBehnken experimental design.
