Chen Dequan, Duggan Chris, Ganley James P, Kooragayala Lakshmana M, Reden Tomas B, Texada Donald E, Langford Marlyn P
Department of Ophthalmology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA.
Protein Expr Purif. 2004 Oct;37(2):426-33. doi: 10.1016/j.pep.2004.06.027.
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.
肠道病毒70型（EV70）的VP1基因含有大量大肠杆菌低使用频率密码子（11.0%）以及位于密码子139侧翼的细菌核糖体结合位点互补序列（RBSCS）5'-UGUCUCCUUUUC-3'。含有编码全长VP1的EV70 cDNA的质粒在大肠杆菌（BL21(DE3)、Rosetta 2(DE3)或Rosetta (DE3)pLysS）中无法表达。在大肠杆菌中重组VP1（rVP1m）的高表达（>总蛋白的8%）需要对编码cDNA进行工程改造（对天然cDNA进行保守修饰），即同时替换位于密码子103和132之间的稀有密码子簇，并替换RBSCS-TCCTTT序列。编码VP1非重叠末端片段N138（1-138个氨基酸）和C170（141-310个氨基酸）的cDNA的稀有密码子频率相似（分别为10.9%和11.2%）。然而，在大肠杆菌中，重组C170（rC170）的高表达不需要对天然cDNA进行修饰，而重组N138（rN138m）的高表达需要对上述稀有密码子簇进行最小程度的同义替换。EV70 VP1基因的稀有密码子簇有五个使用频率最低的精氨酸密码子（AGG/AGA）和三个串联的稀有密码子对（AGGAGG、CUAAGG和AGACUA）。我们的结果表明，EV70 VP1基因中的稀有密码子簇（其本身的稀有密码子排列和/或其相关的mRNA二级结构）和RBSCS，而非稀有密码子频率，构成了抑制其在大肠杆菌中表达的关键因素。
