TY - JOUR
T1 - 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
AU - Klompus, Shelley
AU - Solomon, Gila
AU - Gertler, Arieh
PY - 2008/12
Y1 - 2008/12
N2 - 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.
AB - 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.
KW - Degenerate PCR
KW - MC/9 cells
KW - Protein expression in E. coli
KW - Recombinant human interleukin 10
UR - http://www.scopus.com/inward/record.url?scp=54049123192&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2008.07.013
DO - 10.1016/j.pep.2008.07.013
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C2 - 18725301
AN - SCOPUS:54049123192
SN - 1046-5928
VL - 62
SP - 199
EP - 205
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -