| dc.description.abstract | The goal of this study is to investigate if recombinant porcine trypsinogen can be expressed in inclusion bodies in Escherichia coli (E. coli) cells, refolded and reactivated. The strategy to overexpress a potentially toxic eukaryotic protease in a prokaryotic expression system is to introduce a mutation in the propeptide region of trypsinogen, i.e. Leucine 7 to Proline (L7P). This mutation presumably prevents translocation of the zymogen to the periplasm where it can potentially be degraded by the bacteria’s other proteases. This results in large quantity of the expressed protease in the cytoplasm, leading to inclusion body formation and efficient overexpression of the target protease. Wildtype (WT) and L7P trypsinogen were both transformed into chemically competent E. coli cells. Another trypsinogen construct that also carries the L7P mutation called trypsin+cit was also recombinantly expressed in the same manner as a control. When analyzed by gel electrophoresis, the WT trypsinogen did not show the overexpressed band when induced with IPTG while Both L7P trypsinogen and L7P trypsin showed a strong one. After overexpression, the protease was recovered simply by centrifugation along with extensive washing, and resolubilized using a reversible chemical denaturant. The protease was refolded by dilution into reducing environment. The presence of trypsinogen in resolublized fraction from inclusion bodies and the renatured fraction was proven using gel electrophoresis. The refolded L7P trypsinogen and trypsin+cit were also activated using Enteropeptidase (also known as enterokinase) and commercial trypsin. BSA digestion assay was used to determine the activity of the activated enzymes.
Keywords:
Recombinant protein expression, protein purification, inclusion bodies, protein refolding, trypsinogen, L7P trypsinogen | en_US |
