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

Protein Expression and Purification
Shelley KlompusArieh Gertler

Abstract

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 130K 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 10L of fermentation culture was 60mg and a protocol for its long-term storage as a carrier-free lyophilized powder at -20 degrees was developed.

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Citations

May 28, 2010·Protein Science : a Publication of the Protein Society·Barbara MaertensFrank Schäfer

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