A full-length rat gro cDNA containing the signal sequence was inserted to a plasmid/phage vector pTD-lacs which had the Escherichia coli alkaline phosphatase leader sequence downstream of the lac promoter. After removal of the gro signal sequence by site-directed mutagenesis, the vector was introduced to E. coli JM109. The cells grown in the presence of isopropyl β-D-thiogalactopyranoside were found to contain the recombinant mature rat Gro protein in the periplasmic space. The protein was released from the cells by osmotic shock, and could be purified to homogeneity from the periplasmic fluid by a single-step procedure using reverse phase high performance liquid chromatography. By similar procedures, recombinant human Groα could be obtained. In each case, about 10mg of purified cytokine were obtained from 1 litre of bacterial culture.
- Gro/MGSA/recombinant expression/Neutrophil chemoattractant
ASJC Scopus subject areas
- Immunology and Allergy
- Molecular Biology