Optimization of a Biotechnological Process for Production and Purification of Two Recombinant Proteins: Col G and Col H

Different strategies can be used for increasing production of heterologous recombinant proteins in Escherichia coli. Protein size is often critical for obtaining the best quantity/quality ratio of recombinant protein expression. This study focuses on two recombinant proteins; Class I and class II Collagenases, namely Col G and Col H. Their size is about 150 kDa each. We have developed a method to obtain high levels of cell growth and intracellular expression of each Collagenases in recombinant E. coli BL21(DE3). Batch and Fed-batch fermentation procedures have been performed. Results show that Fed-batch technique was most effective in obtaining the highest cell density for each recombinant bacteria; 28 g/L. We also investigated how to optimize recombinant protein expression; best results were obtained when “multiple shot IPTG induction system” was chosen instead of canonical single shot. By applying a purification protocol based on the use of tangential flow filtration and affinity chromatography we were able to obtain the highest quantity of purified protein: about 13,2 g for Col G and about 12,6 for Col H fermentations. Moreover, by using a stainless steel cooling coil system, we have investigated the effects of low controlled temperature (7°C) during the whole purification process. This system, allowed us to improve the final enzymatic activity of both Collagenases, obtaining 2 fold increase values respect processes performed at room temperature, measured with Pz Grassmann assay. This study shows that, even when the size of a recombinant protein is limiting, is possible to apply a defined Fed-batch protocol to obtain a very high protein production. Moreover these results can be used as a scale up starting step for industrial production and purification of these kind of recombinant enzymes.