Optimized procedures for producing biologically active chemokines

• Published in: Protein expression and purification Vol. 65; no. 2; pp. 251 - 260
• Main Authors: Lu, Quinn, Burns, Matthew C., McDevitt, Patrick J., Graham, Taylor L., Sukman, Abby J., Fornwald, James A., Tang, Xiaoyan, Gallagher, Kathleen T., Hunsberger, Gerald E., Foley, James J., Schmidt, Dulcie B., Kerrigan, John J., Lewis, Tia S., Ames, Robert S., Johanson, Kyung O.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-06-2009
• Subjects: Affinity purification; Chemokines - biosynthesis; Chemokines - isolation & purification; Chemokines - metabolism; Cloning, Molecular; Disulfide bond formation in Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Oxidation-Reduction; Polymerase Chain Reaction; Protein Engineering; Protein Folding; Protein refolding; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Seamless cloning; SUMO hydrolase; SUMO hydrolase; Seamless cloning; Disulfide bond formation in Escherichia coli; Protein refolding; Affinity purification
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2009.01.017

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6×His-SUMO tag in
Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni–NTA–agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6×His-Trx-SUMO tag in an engineered
E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni–NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15
mg/L.