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 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.
Klompus
Shelley
S
Faculty of Agricultural, Food and Environmental Quality Sciences, Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot, Israel.
Solomon
Gila
G
Gertler
Arieh
A
D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D002999:Clone Cells; D004247:DNA / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005612:Freeze Drying; D015723:Gene Library; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017510:Protein Folding; D013816:Thermodynamics
D018076:DNA, Complementary; D016753:Interleukin-10; D004247:DNA