Ligation and Expression of GST-Fused pGEX-KGSP Plasmid

Running head: LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID
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LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID1
Introduction
Expression vectors are widely used as potent molecular tool for obtaining protein
expression in E. Coli cells. pGEX is an expression vector fused with GST coding sequence,
primarily used for ligating gene of interest with the plasmid and screening with glutathione S-
transferase expression. It also consists of Ptac promoter, repressor of lacI, ampicillin
resistance gene (ampR), and multiple cloning site (MCS). Anti-GST-HRP antibodies are
utilized for screening GST expression in cells, confirming successful ligation of the gene of
interest. A leishmania protein is digested with HindIII and EcoR1 to ligate with pGEX-KGSP
plasmid, and immunoblotting was performed followed by SDS-PAGE. Results inferred
successful ligation and expression of gene of interest. Graphical map of pGEX-KGSP
plasmid with circular topology was obtained via Serial Cloner software. Virtual translation of
the ligated expression vector was performed by using Expasy Translate tool. Immunoblotting
was executed using antibody against GST, to confirm successful ligation of the plasmid and
desired protein expression.

LIGATION AND EXPRESSION OF GST-FUSED pGEX-KGSP PLASMID2
Results
Figure 1: