Cloning, expression and purification of CXC chemokine CXCL14
Student name: Ms Nupur Nagar
Guide: Dr Chaithanya Madhurantakam
Year of completion: 2017
Host Organisation: IIT Roorkee, Roorkee
Supervisor (Host Organisation): Dr Krishna Mohan Poluri
Abstract: Chemokines are low molecular weight (~8-10 kDa), alkaline proteins highly specific in chemotaxis,
classified on the basis of position of the cystine residues at N-terminal of the polypeptide as CC, CXC,
CX3C and C chemokines.CXCL14 is a member of CXC chemokine family. It is a small chemotactic protein
having functional role in homeostatic as well as immune responses. Structurally conserved, it is a non-ELR
chemokine having characteristic five amino acid residues at N-terminal end for its own degradation. Beside
chemotaxis, CXCL14 has been known to act as an antimicrobial peptide and also has antitumor activity which
makes it of great importance. Owing to its diverse functional properties, hCXCL14 was cloned in pET32 Xa/LIC
vector by traditional cloning method resulting in a fusion gene construct. The clone was overexpressed in E. coli
BL21 strain and was observed to be found entirely as inclusion bodies. Two different protocols using 6M GdnHCl
and 8M urea were used to isolate and purify functional hCXCL14 from the insoluble form. Further it was observed
that 6 M GdnHCl with dilution method yield more protein over 8M urea refolding method.
Keywords: chemokine, antimicrobial, protein refolding, purification, cloning.
