Optimization of Soluble Expression of Chimeric Enzyme of Thermolysin and Elastase by Co-expressing the Mature protein and Prosegment

Thermolysin, a thermostable neutral zinc metalloproteinase derived from Bacillus thermoproteolyticus, is synthesized as inactive pre-proenzyme. The pro-sequence in the prothermolysin acts as a molecular chaperone leading to an autocleavage of the peptide bond linking the pro- and mature sequences. This protein represents low stability againts organic solvents, limiting its application in peptide synthesis. On the other hand, elastase achieved from Pseudomonas aeuroginosa, is extremely stable Zn-metalloprotease in organic solvents. To achieve a thermostable and organic solvent stable variant of thermolysin, we have previously engineered a chimeric enzyme in which 64 residues in the N-terminal domain of thermolysin was replaced with equivalent 65 residues of elastase. In this study, we designed and used a new expression strategy to decrease the production of inclusion bodies and soluble active proteins. The mature and prosegment as a chaperone of thermolysin containing N-terminal pelB leader sequence were co-expressed in E. coli as independent polypeptides under the T7 promoter in pETDuet-1 vector. N-terminal His-tag and cleavage site of TEV protease was used for protein purification and remove His-tag respectively. The PelB signal sequence was inserted in the beginning of the gene in order to protein secretion into the periplasmic region. A series of experiments was conducted to obtain optimal protein expression condition. Osmotic shock method was used to achieve periplasmic soluble expression and the target protein was measured in periplasmic region, cytoplasm, and culture medium. SDS-PAGE showed a band of 35 kDa for preplasmic fraction and the enzymatic hydrolysis of casein indicated an improvement in production of soluble and active protein compared to the latest study. Therefore, it seems that this new co-expression method is more sufficient than the previous method for production of chimeric enzyme.