Expression optimization of ⍺-ketoglutarate dependent non-heme Fe(II) dioxygenases containing non-canonical amino acid in the facial triad

master's thesis

• Title (croatian): Optimizacija ekspresije α-ketoglutarat-ovisnih Fe(II) hem-neovisnih dioksigenaza s neprirodnom aminokiselinom u aktivnom mjestu
• Author: Josipa Smoljo
• Mentor: Anita Slavica (mentor)
• Committee member: Renata Teparić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2021-04-30, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Expression of two L-leucine 5-hydroxylases, one isolated from Anabaena variabilis ATCC 29413 (AvLDO_wt) and second from Streptomyces muensis DSM 9461 (GriE_wt), were studied in two expression systems - pET28a(+) and pASKGATE. In vitro cloning methods FastCloning and Gateway® technology were performed in order to clone the genes in different vectors. Furthermore, site-directed mutagenesis QuikChange™ was done to clone the amber codon to the genetic code of the wild types at previously located sites, necessary for N-methylhistidine incorporation in the facial triad of the enzymes by the stop codon suppression methodology. The successful construction of these rationally designed enzymes was one of the rare examples of the incorporation of non-canonical amino acid in general, and N-methylhistidine specifically, in the active site of non-heme dioxygenases. Furthermore, the soluble expression of AvLDO and GriE (both wild types and mutants) was investigated using different concentrations of inducers and supplemented N-methylhistidine and successfully improved as observed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Accordingly, the substrate scope of the GriE wild type and mutant was studied and assessed towards L-leucine and L-isoleucine by thin-layer chromatography.

Abstract (english)

Ekspresija dviju L-leucin 5-hidroksilaza, jedne izolirane iz Anabaena variabilis ATCC 29413 (naziva AvLDO_wt) te druge izolirane iz Streptomyces muensis DSM 9461 (GriE_wt), proučavana je u dva plazmida - pET28a (+) i pASKGATE. In vitro metode kloniranja, FastCloning i Gateway® tehnologija primijenjene su zbog kloniranja gena u različite vektore. Nadalje, mjesno specifična mutageneza QuikChange™ primijenjena je kako bi se klonirao stop kodon na prethodno odabrane pozicije u genima AvLDO_wt i GriE_wt, što je neophodno za ugradnju N-metilhistidina u aktivno mjesto ovih enzima pomoću metodologije stop kodon supresije. Uspješna konstrukcija ovih racionalno dizajniranih enzima jedan je od rijetkih primjera ugradnje neprirodne aminokiseline općenito, a posebno N-metilhistidina, u aktivno mjesto hem-neovisnih dioksigenaza. Nadalje, istražena je ekspresija AvLDO i GriE (divljih tipova i mutanata) u topljivoj frakciji korištenjem različitih koncentracija induktora i N-metilhistidina te je uspješno poboljšana, kako je i potvrđeno poliakrilamid gel elektroforezom u prisutnosti natrijeva dodecil-sulfata. Također, tankoslojnom kromatografijom je proučavana aktivnost GriE divljeg tipa i mutanta, prema supstratima L-leucinu i L-izoleucinu.

• Keywords: L-leucine 5-hydroxylases; AvLDO; GriE; stop codon suppression; N-methylhistidine
• Keywords (english): L-leucin 5-hidroksilaze; AvLDO; GriE; stop kodon supresija; N-methilhistidin
• Language: english
• URN:NBN: urn:nbn:hr:159:499801
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-06-08 11:56:30