SYNTHESIS AND EXPRESSION IN ESCHERICHIA-COLI OF A HUMAN NEUTROPHIL-ACTIVATING PROTEIN-1 INTERLEUKIN-8 GENE

The complete gene coding for human neutrophil activating protein-1/int erleukin-8 was synthesized using a semi-chemical semi-enzymatic method . The synthetic gene was then overexpressed in Eseherichia coli under the temperature-regulated control of the P(R)P(L) tandem promoters. As determined by SDS-PAGE and densitometry, the overexpressed protein co mprised up to 18.5% and 10.9% of the total soluble protein in E. coli cells grown in shake flasks and in batch fermentation, respectively. T he recombinant NAP-1/IL-8 was then purified to >95% homogeneity by gel filtration and cation exchange chromatography. The purified protein a ppeared as a single band on the SDS-PAGE gel and possessed potent chem otactic activity in the concentration of <10 ng/ml, as assayed by the agarose plate method. An early skin reactivity was also observed when the pure NAP-1/IL-8 was injected subcutaneously into the rabbits. The N-terminal 36 amino acid sequence of the recombinant NAP-1/IL-8 was de termined using the Edman method and was shown to be identical to that of the ntive protein.