Identification and characterization of a +1 frameshift observed during the expression of Epstein-Barr virus IL-10 in Escherichia coli.
Epstein-Barr virus IL-10 (ebvIL-10) mimics the biological functions of cellular IL-10 including a number of immunoinhibitory activities on diverse immune cells. Characterization of ebvIL-10 and several mutants, expressed in Escherichia coli, by gel filtration chromatography and mass spectrometry revealed a +1 frameshift upon ebvIL-10 expression. The frameshift is caused by the rare AGG codon at ebvIL-10 Arg159, which is followed by the most inefficient stop signal, UGAC. The frameshift was corrected by substituting the rare AGG codon with an abundant arginine codon, CGU, or by enhancing the level of tRNA that decodes the AGG codon. As a result, ebvIL-10 expression levels increased by approximately 3-fold and the purity of the protein improved from 85-95% to 98-99%. The correction of the frameshift has been essential for continuing structural and biophysical studies of ebvIL-10.
Yoon
Sung Il
SI
Department of Microbiology and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Walter
Mark R
MR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003062:Codon; D018388:Codon, Terminator; D018076:DNA, Complementary; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D016368:Frameshift Mutation; D015870:Gene Expression; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D016753:Interleukin-10 / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012347:RNA, Transfer, Arg / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000494:pharmacology
D003062:Codon; D018388:Codon, Terminator; D018076:DNA, Complementary; D010446:Peptide Fragments; D012347:RNA, Transfer, Arg; D016753:Interleukin-10; D014357:Trypsin