Molecular verification and bioinformatic analysis of a serine protease gene from Bacillus Pumilus

Abstract

Enzymatic wound debridement is a promising effective approach; however, there is a limited number of enzymes that can be used for debridement, and many existing enzymes in the market can cause side effects. Cardiovascular diseases are the main causes of death and disability. It is usually associated with an increased risk of blood clots. The current medications are limited in number and have serious bleeding risks. This study is focused on the verification of the previously cloned serine protease gene via PCR, sequencing the purified plasmid, and analyzing its amino acid sequence through different bioinformatics tools. Eventually, this protease is concluded to have the potential to be a new wound debridement and clot-dissolving agent that can be more affordable, effective, with a better side effects profile. The serine protease gene from Bacillus pumilus was previously cloned into pET-21b expression vector and transformed in E. coli HST08. The vector was successfully extracted from the host bacteria and quantified by Nanodrop spectrophotometer to record a concentration of 92.9 ng/μl, with acceptable 260/280 and 260/230 ratios; 1.85, 2.22 respectively. The purified plasmid was then used as a DNA template and subjected to amplification via PCR. The amplified gene resolved in the agarose gel and was found to constitute approximately 1077 bp. The sequencing result was showed 100% identity to serine protease from Bacillus pumilus (Accession No: kll00441.1). A bioinformatics study was conducted to generate fundamental data that will give some insights into the protein nature and structure. Cytoplasmic, transmembrane, and non-cytoplasmic regions were predicted by the Phobius online tools. ProtParam tool computed the molecular weight of the protease to be 37.8 kilodaltons, acidic in nature, moderately hydrophilic, and have a good stability index. SMART online tool predicted that the serine protease contains two highly conserved domains; trypsin-like peptidase, and PDZ domains. The 3D model of the serine protease was generated based on the HtrA protease Deg1 structure through the Swiss model server. The proposed model passes all three quality validation methods (Errat, Verify 3D, and Procheck). The three catalytic residues at the active site of the enzyme are found to be His91, Asp121, and Ser202. Additionally, the generated model was superimposed to the human tissue plasminogen activator (Currently used thrombolytic agent) to propose a structure-function relationship. The superimposition between the two structures was good, and the catalytic triad has a good alignment. To conclude, the serine protease was found to be a good candidate as a potential fibrinolytic agent as its sequence was verified successfully with the reference gene, and the generated bioinformatic data have shown it can be expressed and purified successfully for further characterization.