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2014•Unpublished venueRequires access

The Cloning and Prokaryotic Expression ofδ-aminolevulinic acid(ALAS)Gene

Zhao Yan-qion

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Abstract

To express and purifyδ-aminolevulinic acid(ALAS)in E.coli,liver RNA was reverse transcribed into cDNA.The ALAS gene was amplified by PCR and confirmed by sequencing.The pET30a(+)-ALAS recombinant vector was successfully constructed.The recombinant vector was transformed into Rosetta(DE3)and induced by IPTG.SDS-PAGE analysis showed that the fusion protein was about 70 kDa,and a small portion of the protein was expressed in the supernatant.The expressed His6-tagged protein was purified by Ni 2+affinity chromatography column and characterized by SDS-PAGE and confirmed by western blot analysis.

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What this paper is about

To express and purifyδ-aminolevulinic acid(ALAS)in E.coli,liver RNA was reverse transcribed into cDNA.The ALAS gene was amplified by PCR and confirmed by sequencing.The pET30a(+)-ALAS recombinant vector was successfully constructed.The recombinant vector was transformed into Rosetta(DE3)and induced by IPTG.SDS-PAGE analysis showed that the fusion protein was about 70 kDa,and a small portion of the protein was expressed in the supernatant.The expressed His6-tagged protein was purified by Ni 2+affinity chromatography column and characterized by SDS-PAGE and confirmed by western blot analysis.

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Available abstract

To express and purifyδ-aminolevulinic acid(ALAS)in E.coli,liver RNA was reverse transcribed into cDNA.The ALAS gene was amplified by PCR and confirmed by sequencing.The pET30a(+)-ALAS recombinant vector was successfully constructed.The recombinant vector was transformed into Rosetta(DE3)and induced by IPTG.SDS-PAGE analysis showed that the fusion protein was about 70 kDa,and a small portion of the protein was expressed in the supernatant.The expressed His6-tagged protein was purified by Ni 2+affinity chromatography column and characterized by SDS-PAGE and confirmed by western blot analysis.

Key concepts: Recombinant DNA, lac operon, Molecular biology, Fusion protein, Complementary DNA, Affinity chromatography, Cloning (programming), Gene

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