Klonování, exprese a purifikace vybraného mykobakteriálního enzymu

• Main Author: Švidrnochová, Michaela
• Other Authors: Novotná, Eva
• Format: Dissertation
• Language: Czech
• Published: 2013
• Online Access: http://www.nusl.cz/ntk/nusl-324160

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Michaela Švidrnochová Supervisor: Mgr. Eva Novotná, Ph.D. Title of diploma thesis: Cloning, expression and purification of selected mycobacterial enzyme Mycobacterium tuberculosis produces many enzymes which are nescessary for its growing and living. Inhibition of enzymes participating in biosynthesis are a potential target of new drugs in treatement of tuberculosis. Naphtoate synthase is an mycobacterial enzyme in biosynthetic pathway of menaquinone. It has a crucial meaning in electron transport chain under anaerobic conditions. Naphtoate synthase belongs to crotonase superfamily, catalyses a conversion of o-succinylbenzoyl-CoA (OSB-CoA) into 1,4-dihydroxy-2-naphtoyl-CoA (DHNA-CoA). Plasmid pET-28b(+) and a segment of DNA encoding the sequence of menB amplified by polymerase chain reaction was used for preparation of recombinant protein menB. Ligated plasmid was transformed into the cells E. coli HB101 by heat shock. Expression cells E. colli BL21 were used for the expression and the expression itself was started by the addition of IPTG. Prepared protein was isolated and purified by a machine Äkta which used the method of affinity chromatography.