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Bioinformatics analysis and molecular cloning of an extracellular serine protease from acinetobacter baumannii

Lim, Aik Siang (2018) Bioinformatics analysis and molecular cloning of an extracellular serine protease from acinetobacter baumannii. Masters thesis, Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering.

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Abstract

Drug resistant Acinetobacter baumannii topped the list for antibiotic resistant ‘critical’ pathogens that was released by the World Health Organisation (WHO) in February 2017. The list was intended to guide and promote research and development (R&D) of antibiotics. One of the factors that may contribute to A. baumannii virulence are the secretory proteases that this bacteria produces. In order to design effective antibiotocs and treatment targeting secretory proteases from A. baumannii, the gene coding for the proteases needed to be cloned to produce recombinant form of the proteins that can be easily expressed and purified in the quantities and purity suitable for functional and structural studies. A secreted serine protease was identified from A. baumannii, termed as “SPSFQ”. Bioinformatics analysis using BLAST and multiple sequence alignment indicated that the enzyme belonged to serine endopeptidases (E.C. 3.4.21.-) family with a predicted catalytic triad motif of D130/H163/S315. Structure of SPSFQ modelled using the homology modeling software, I-TASSER revealed that the enzyme folding was highly conserved to keratinase 5WSL with seven stranded parallel β sheets flanking by six α helices and four β sheets made of two anti-parallel strands. SPSFQ with 1104 bp coding for 368 amino acids was subcloned into pET-22b(+) between BamH1 and Sal1 and expressed in periplasmic fraction of E. coli BL21 (DE3). Total cell protein with induction condition at 16 ºC and 25 ºC with 1mM IPTG showed two distinct bands around 40 kDa (proenzyme form) and 30 kDa (active form) in western blot. Cell lysate did not show any activity during enzymatic assay probably because of SPSFQ was expressed in inclusion form. As a conclusion, SPSFQ was successfully sub-cloned and expressed in E. coli BL21 (DE3). Further study will focus on purification and characterization of SPSFQ in order to identify the cellular importance of SPSFQ towards A. baumannii.

Item Type:Thesis (Masters)
Additional Information:Thesis (Sarjana Sains, Pengkhususan: Bioteknologi) - Universiti Teknologi Malaysia, 2018
Subjects:Q Science > QH Natural history > QH301 Biology
Divisions:Biosciences and Medical Engineering
ID Code:78678
Deposited By: Fazli Masari
Deposited On:29 Aug 2018 15:35
Last Modified:29 Aug 2018 15:35

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