New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

TY - JOUR

T1 - New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

AU - Tufail, Afzaal

AU - Akkad, Saeed

AU - Noble, Amanda R.

AU - Fascione, Martin A.

AU - Signoret, Nathalie

N1 - Publisher Copyright: © The Author(s) 2024.

PY - 2024/10/15

Y1 - 2024/10/15

N2 - Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.

AB - Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.

KW - CCL5

KW - CCR5

KW - Chemokine activity testing

KW - Protein purification

KW - Recombinant chemokine production

UR - http://www.scopus.com/inward/record.url?scp=85206528061&partnerID=8YFLogxK

U2 - 10.1038/s41598-024-75327-y

DO - 10.1038/s41598-024-75327-y

M3 - Article

C2 - 39406925

AN - SCOPUS:85206528061

SN - 2045-2322

VL - 14

JO - Scientific reports

JF - Scientific reports

IS - 1

M1 - 24188

ER -