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. 2003 Feb;185(3):1045–1058. doi: 10.1128/JB.185.3.1045-1058.2003

FIG. 5.

FIG. 5.

Overproduction of pTiA6 VirB4 in E. coli strain BL21. Heterologous overexpression of virB4 in E. coli is inhibited by different codon usage. The virB4 gene contains 11 codons for arginine, nine codons for leucine, and two codons for proline, which are rare in E. coli. Plasmid pACYC-RIL encodes the tRNAs argU (AGA, AGG), ileY (AUA), and leuW (CUA). Freshly transformed strains BL21(pCR6) and BL21(pCR6, pACYC-RIL) were induced with 1 mM IPTG and grown for 5 h. Crude cell extracts of induced and noninduced cells were separated on an SDS-15% polyacrylamide gel and stained with Coomassie blue. Lane a, molecular mass standard; lane b, noninduced BL21(pCR6); lane c, induced BL21(pCR6); lane d, noninduced BL21(pCR6, pACYC-RIL); lane e, induced BL21(pCR6, pACYC-RIL). The arrow in lane c indicates the faint band of VirB4 resulting from induction in the absence of pACYC-RIL; the prominent band in lane e was shown to be VirB4.