During inflammation, leukocyte trafficking to the injury site is regulated by a group of pro-inflammatory proteins called chemokines. While essential, excessive leukocyte trafficking leads to inflammatory diseases. Neutralising chemokines seems to be a logical route to developing anti-inflammatory therapeutics, but is challenging due to promiscuous binding of chemokines to their receptors. Developing a molecule that can bind to multiple chemokines would offer a better solution.
Ticks produce proteins called evasins that inhibit multiple chemokines. Discovered by our lab, the evasin EVA-AAM1001 (or EVA-A) binds to 20 different chemokines. Although promising, most of the EVA-A population is aggregated during protein purification, likely due to misfolding events. Our project aims to combinatorially engineer EVA-A to obtain a more stable mutant, using the phage display method. We have successfully displayed the wild-type EVA-A on the surface of M13 phages and shown binding to the chemokine CCL16. We have designed a combinatorial library of EVA-A by mutating its hydrophobic core. We will display this library on the surface of the M13 phages and select using CCL2, CCL7 and CCL8 which are implicated in aggravating inflammation in various diseases like atherosclerosis, pulmonary fibrosis etc. After enrichment and selection of stable mutants, we will use next-generation sequencing to reveal the required mutations conferring the stability of EVA-A.