Expression of enterovirus 70 capsid protein VP1 in Escherichia coli.
The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS). High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli.
Chen
Dequan
D
Department of Ophthalmology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA. dchen@lsuhsc.edu
Duggan
Chris
C
Ganley
James P
JP
Kooragayala
Lakshmana M
LM
Reden
Tomas B
TB
Texada
Donald E
DE
Langford
Marlyn P
MP
D001665:Binding Sites; D015153:Blotting, Western; D036022:Capsid Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D003062:Codon; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004770:Enterovirus / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
D036022:Capsid Proteins; D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins