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

摘要

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