Cloning of human survivin in pET-28a and its protein expression study in E.coli BL21(DE3)

Tirmomenin, Mahsa; Ataei, Farangis; Hosseinkhani, Saman

Cloning of human survivin in pET-28a and its protein expression study in E.coli BL21(DE3)

Document Type : Original Research

Authors

Mahsa Tirmomenin ¹

Farangis Ataei ²

Saman Hosseinkhani ¹

¹ tarbiat modares university

² Tarbiat modares university

Abstract

Inhibitor of apoptosis (IAP) are a family of proteins that block cell death through caspase activity. Survivin is smallest IAPs family member that overexpresses in different cancer types but not in normal tissue except embryonic tissue. Survivin may be used as a new marker to stratify cancer patients for more optimal treatment modalities. The aim of the current study was to investigate survivin DNA cloning into pET-28a and its expression in E.coli.

The sequence of survivin gene was amplified by PCR using specific primers and pcDNA-survivin temple. PCR product and pET-28a plasmid were digested by HindIII/NheI restriction enzymes and survivin was ligated into the digested vector. Then, the ligation product was transformed into the E.coli DH5a competent cells and screened by antibiotic selection marker (kanamycin). Positive colonies were selected by colony PCR and screened by double digestion of isolated plasmid. One positive colony was sequenced and confirmed. The recombinant plasmid was transformed into the expression strain of E.coli (BL21) by chemical method. The expression of survivin was induced in the different conditions and expression level investigated by SDS-PAGE.

The size of PCR product in agarose gel showed the correctness of amplification. The digested pET-28a plasmid also indicated the correctness of enzymatic reaction. The sequence of the cloned fragment revealed a 100% similarity to the human survivin. In expressing, adding IPTG increased the expression of survivin protein in all conditions, especially 37 ᵒC from 2 h after induction. At all conditions, most of survivin accumulated in the bacteria as inclusion body.

Keywords

Survivin

PCR

Cloning

pET-28a

BL21(DE3)

20.1001.1.23222115.1399.11.3.8.1

Subjects

Molecular biotechnology

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