Abstract
Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.
Highlights
Chemokines are key players in the recruitment and retention of leukocytes at tissue locations and function by activating specific G protein-coupled receptors (GPCRs) [1]
ubiquitin like protease-1 (ULP1) was dialysed separately into reducing agent free buffer to ensure CXCL8 would not be reduced upon digestion
We present an efficient protocol for expressing soluble CXCL8 as a SUMO fusion in E. coli Shuffle T7 Express
Summary
Chemokines are key players in the recruitment and retention of leukocytes at tissue locations and function by activating specific G protein-coupled receptors (GPCRs) [1]. Recombinant chemokine production has been key to these efforts but is not without challenges. Methods that focus on chemical synthesis or expression in E. coli typically yield unfolded, reduced, and inactive chemokines, necessitating refolding steps that can be time consuming and challenging to optimise. A signal peptide of around 20 amino acids is typically cleaved to liberate the mature chemokine with full agonist activity. This process has to be mimicked in prokaryotic expression systems
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