A simple novel method for the preparation of noncovalent homodimeric, biologically active human interleukin 10 in Escherichia coli—Enhancing protein expression by degenerate PCR of 5′ DNA in the open reading frame

DNA inserts encoding human interleukin 10 (hIL-10), optimized for codon usage and secondary RNA structure, were purchased from several commercial sources and subcloned into a pMon vector. Despite the optimization, protein expression was nil. We therefore subjected the 5′ segment of the cDNA encoding N-terminal amino acids 2–11 to degenerate PCR in order to create a small library of 130 K theoretical cDNA combinations that would not change the respective amino acid sequence and tested their expression. After screening over 320 colonies 10 hIL-10 clones encoding the original amino acid sequence were identified. Three nucleotide substitutions were sufficient to ensure reasonable protein expression. Subsequently, hIL-10 was expressed in Escherichia coli, refolded and purified to homogeneity, yielding over 95% electrophoretically pure noncovalent homodimeric protein, which was biologically active in MC/9 cells. The yield of recombinant hIL-10 from 10 L of fermentation culture was 60 mg and a protocol for its long-term storage as a carrier-free lyophilized powder at −20° was developed.