EXPRESSION OF ROSSELLOMOREA AQUIMARIS MKSC 6.2 SORTASE D-ENCODING GENE IN ESCHERICHIA COLI

• Main Author: Adilah, Nurjihan
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/82779
• Institution: Institut Teknologi Bandung

Sortase is a cysteine protease and transpeptidase that catalyzes the covalent assembly and anchoring of surface proteins to the peptidoglycan cell wall of Gram-positive bacteria. Sortase D is involved in sporulation in Bacillus anthracis. The nucleotide sequence of the sortase D gene (srtD) originating from the marine bacterium Rossellomorea aquimaris MKSC 6.2 (RaqsrtD) was previously determined to be 540 base pairs long. Since the expression of this protein-coding gene has not been conducted, the aim of this study was to construct a recombinant plasmid pET-16b-srtD and express srtD from R. aquimaris MKSC 6.2 in Escherichia coli. The research began with primer design based on the nucleotide sequence of RaqsrtD, with added XhoI and BamHI enzyme cleavage sites. The srtD gene was amplified via PCR using the pGEM-T Easy-RaqsrtD plasmid template, with an optimal annealing temperature of 46.4 ?. The resulting PCR fragments were then inserted into the pGEM-T Easy cloning vector. Competent E. coli TOP10F' cells were transformed with the recombinant plasmid pGEM-T Easy-srtD. The target pGEM-T Easy-srtD recombinant plasmid was subsequently restricted to isolate srtD and inserted into the pET-16b expression vector. E. coli TOP10F' cells were transformed with the recombinant plasmid pET-16b-srtD for amplification. The transformant cells were characterized by restriction analysis, colony PCR, and nucleotide sequencing using the Sanger method. The desired recombinant plasmid pET-16b-srtD was used to transform E. coli BL21(DE3). Expression of srtD was carried out, and Sortase D was produced as a soluble protein in E. coli BL21(DE3) with a size of ~28.3 kDa under optimal production conditions using Luria Bertani growth medium, 0,1 mM IPTG as inducer, and incubation at room temperature for 3 hours. The soluble Sortase D was subsequently purified and concentrated, resulting in a concentration of 142 ?g/mL. Peptide mapping by LC-MS/MS to determine the protein sequence showed that the generated sequence matched the theoretical sequence with a coverage of 99.51%. Alignment results indicated that this sequence corresponds to Sortase D, which is most closely related to Bacillus sp. Marseille-Q1617 and R. oryzaecorticis.