Kloniranje i ekspresija gena za SGNH-hidrolazu iz bakterije Streptomyces coelicolor u heterolognom sustavu

master's thesis

• Title (english): Cloning and expression of SGNH-hydrolase of Streptomyces coelicolor in heterologous host
• Author: Marija Marić
• Mentor: Dušica Vujaklija (mentor)
• Mentor: Ivana Ivančić Baće (sumentor)
• Committee member: Dijana Škorić (predsjednik povjerenstva)
• Committee member: Dubravka Hranilović (član povjerenstva)
• Committee member: Nataša Bauer (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2009, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Bakterijske SGNH-hidrolaze su značajna grupa enzima koja još uvijek nije dobro istražena, ni strukturalno ni biokemijski. Izvanstanična lipaza iz obitelji SGNH-hidrolaza iz bakterije Streptomyces coelicolor soja A3(2) (Q93J06, SCO7513) je djelomično biokemijski okarakterizirana. Cilj ovog istraživanja bio je izrada konstrukta gena s dodatkom kodona za 6 histidina na 3' kraju gena te kloniranje i ekspresija gena u bakteriji Streptomyces lividans soja TK23 sa svrhom uspostavljanja učinkovite i brze metode za pročišćavanje proteina. Nakon ekspresije rekombinantnog gena u heterolognom sustavu protein je pročišćen taloženjem s amonijevim sulfatom i afinitetnom kromatografijom na nosaču s imobiliziranim metalom. Prisustvo pročišćenog proteina je dokazano Western analizom, a uočeno je da tijekom pročišćavanja dolazi do gubitka specifične aktivnosti. Iako je prema literaturi ovaj protein označen kao izvanstanični, analiza softverom SignalP 3.0 za predviđanje signalnog slijeda proteina nije prepoznala signalni slijed u SGNH-hidrolazi, što upućuje na mogućnost zadržavanja proteina unutar stanica.

Abstract (english)

Bacterial SGNH-hydrolases are an important group of enzymes which are still not well characterised structurally or biochemically. Extracellular SGNH-hydrolase from Streptomyces coelicolor A3(2) (Q93J06, SCO7513) was earlier partially biochemically described. Aim of this investigation was to construct a gene with an additional codons for 6 histidines on 3' end of the gene. Cloning and expression of the gene Streptomyces lividans TK23 was performed with a purpose of establishing an efficient and quick protein purification method. After the expression of the recombinant gene in heterologous system protein was purified applying ammonium sulfate precipitation and immobilized-metal affinity chromatography. Presence of the purified protein was confirmed with Western analysis. The protein lost specific activity during the purification. Although according to the literature this protein was annotated as extracellular, software SignalP 3.0 failed to recognize a signal sequence in SGNH-hydrolase which implicates a possibility that the protein retains inside the cell.

• Keywords: Streptomyces coelicolor; GDSL/SGNH obitelj; lipaza
• Keywords (english): Streptomyces coelicolor; GDSL/SGNH family; lipase
• Language: croatian
• URN:NBN: urn:nbn:hr:217:936343
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2018-03-13 11:40:57