OPTIMIZED GENE SYNTHESIS, HIGH-LEVEL EXPRESSION, ISOTOPIC ENRICHMENT,AND REFOLDING OF HUMAN INTERLEUKIN-5

Structural studies on soluble proteins using nuclear magnetic resonanc e (NMR) spectroscopy and other structural methods in general require l arge quantities of isotopically enriched proteins. Human interleukin-5 is a disulfide-linked homodimeric cytokine implicated in asthmatic re sponse. The development of a high yield overexpression system for huma n interleukin-5 is an important prerequisite to using modern multidime nsional NMR in the characterization of the solution structure of the p rotein and to characterize interactions with a soluble receptor domain . Significant amounts of the protein were expressed using an optimized synthetic gene in a high yield expression system. Gene synthesis was accomplished through the ligation of six oligonucleotides composed of optimized codons. The ligated fragments were further amplified by a po lymerase chain reaction and then subcloned into the T7 RNA polymerase based overexpression vector pET11a. However, the induced protein accum ulated in the form of inclusion bodies. Initially, the protein was sol ubilized under denaturing conditions and purified in these denaturing conditions by passage through a single S-200 HR sizing column. Finally , protein refolding was initiated in the presence of 2 M urea followed by dialysis. This protocol yielded 40 mg of biologically active, isot ope-enriched protein from 4 liters of minimal medium thus facilitating structural studies by NMR. The strategy described may be of immense v alue in the production of significant quantities of recombinant, eukar yotic proteins for structural and other studies. (C) 1997 Academic Pre ss.