Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli.
In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E. coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E. coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNA(CGG) levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E. coli, allowing for efficient and accurate translation of heterologous gene products.
McNulty
Dean E
DE
Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA. dean_e_mcnulty@gsk.com
Claffee
Brett A
BA
Huddleston
Michael J
MJ
Porter
M Lauren
ML
Cavnar
Katie M
KM
Kane
James F
JF
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003062:Codon; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D016368:Frameshift Mutation; D005822:Genetic Vectors; D005973:Glutamine / Q000737:chemistry; D018258:Herpesvirus 2, Human / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D003062:Codon; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005973:Glutamine; D012343:RNA, Transfer; D001120:Arginine