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.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