Akademik Veri Yönetim Sistemi
JOURNAL OF PURE AND APPLIED MICROBIOLOGY, cilt.18, ss.1326-1335, 2024 (ESCI)
Proteins are essential for the proper functioning of cells. The techniques of cloning and protein production have facilitated the advancement of various fields and the creation of specific proteins for industrial and therapeutic uses. The bacterium Aeribacillus pallidus, which is able to survive in extreme conditions, is being studied with a view to identifying its robust enzymes. The objective of this study was to clone the protease gene from the A. pallidus P18 strain into the SUMO vector and produce recombinant protein in Escherichia coli BL21 for protein production. The protease enzyme gene from the A. pallidus P18 strain was isolated and amplified by using PCR. The PCR product was transferred into the SUMO expression vector and amplified in One Shot (R) Mach1TM-T1R bacteria, followed by colony PCR. Plasmid isolation was performed after positive colony selection. Gene integration was confirmed by cross-PCR using the gene forward, and vector reverse primers. For expression, the plasmid was transferred to E. coli BL21 cells. Two cultures were induced with different IPTG concentrations (0.5 mM and 1 mM) to optimize protein production. Bacterial cells were lysed, and SDS-PAGE analysis was conducted. Purification involved cell lysate preparation and purification using a ProbondTM column. SDSPAGE and Coomassie Brilliant Blue G-250 staining confirmed successful purification. The results of this study indicate that the optimal product for protein production is that derived from a culture induced with 1 mM IPTG. Upon completion of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure, the weight mass of the produced protein was determined to be 37 kDa, as indicated by the result of the gel stained with Coomassie brilliant blue G-250. This research successfully cloned the protease enzyme gene from the A. pallidus P18 strain using the pET-SUMO vector, performed purification and achieved the targeted result of protein production.