Cloning and Expression of Fusion Protein Human Beta-Defensin 2 Green Fluorescent Protein in Escherichia coli

Pedersen, Kasper

4. term

Nanotechnology, Master

2013

2013-09-10

94 pages

I dette kandidatspeciale blev opløseligt HbD-2-turboGFP fusionprotein udtrykt i E. coli Origami 2(DE3). Fluorescensmålinger af fusion proteinet indikerede en korrekt foldet og aktiv turboGFP fusions-partner. Fluorescensmålinger viste også en meget højere proteinkoncentration i bakterier groet under IPTG induktion, end i den ikke-inducerede reference. Fusionproteinets størrelse blev bekræftet på en SDS-PAGE gel. pET-11a-DEFB4A-GFP vektoren blev konstrueret ved ligation af DEFB4A-GFP og pET-11a, men en normal sub-kloning fremgangsmåde blev ikke benyttet, da DEFB4A-GFP genet har en - på genet - NdeI restriktionssekvens. Således blev en NdeI partiel restriktion af DEFB4A-GFP først udført, men denne var ikke succesfuld. Derfor blev en PCR punktmutation udført for at mutere den - på genet - NdeI restriktionssekvens fra CATATG til CATTTG. Dette var succesfuldt, og det muterede gen kunne blive klippet med NdeI og BamHI restriktionsenzymer og blive ligeret med klippet pET-11a for at forme pET-11a-mut-DEFB4A-GFP vektor. Sekventeringsdata af den konstruerede vektor viste ingen mutationer andre end den producerede punktmutation.

In this master’s thesis, soluble HbD-2-turboGFP fusion protein was expressed in E. coli Origami 2(DE3). Fluorescence measurements of the fusion protein indicated a correctly folded and active turboGFP fusion-partner. Fluorescence measurements also showed a much higher protein concentration in bacteria grown under IPTG induction, than in the uninduced reference. The fusion protein size was confirmed on an SDS-PAGE gel. The pET-11a-DEFB4A-GFP vector was constructed by ligation of DEFB4A-GFP and pET-11a, however, normal subcloning strategies were not performed, as the DEFB4A-GFP gene had an on-gene NdeI restriction site. Thus, an NdeI partial digestion of DEFB4A-GFP was first carried out, but this was unsuccessful. Then a PCR site-directed point mutation was performed to mutate the on-gene NdeI restriction sequence from CATATG to CATTTG. This was successful, and the mutated gene could be digested with NdeI and BamHI restriction enzymes and ligated into digested pET-11a to form pET-11a-mut-DEFB4A-GFP vector. Sequencing data of the constructed vector showed no mutations other than the point-mutation produced.

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