کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2021239 | 1069236 | 2009 | 10 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Optimized procedures for producing biologically active chemokines Optimized procedures for producing biologically active chemokines](/preview/png/2021239.png)
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.
Journal: Protein Expression and Purification - Volume 65, Issue 2, June 2009, Pages 251–260