Tomich C S, Olson E R, Olsen M K, Kaytes P S, Rockenbach S K, Hatzenbuhler N T
Molecular Biology Research, Upjohn Company, Kalamazoo, MI 49007.
Nucleic Acids Res. 1989 Apr 25;17(8):3179-97. doi: 10.1093/nar/17.8.3179.
We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E. coli. The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E. coli using a pBR322-derived vector. However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product. Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA. There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation. To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out. The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation. Removal of this stem-loop results in a 100-fold increase in BSt expression. However, the expression level is still low, amounting to only 0.5-1% of total cell protein. High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons. These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation.
我们研究了牛生长激素（BSt）的表达，以更深入了解影响大肠杆菌中翻译的各种因素。使用源自pBR322的载体，编码成熟牛生长激素的未修饰cDNA在大肠杆菌中不会产生大量的BSt。然而，在BSt cDNA前与来自trpLE的16个密码子进行翻译融合，融合产物占总细胞蛋白的20%以上。通过测量mRNA的速率和完整性来分析转录，证实转录后事件是导致BSt cDNA表达不佳的原因。mRNA的5'区域存在两个潜在的茎环结构，可能会干扰翻译。为了研究它们对翻译的影响，进行了lacZ融合和寡核苷酸诱变。结果表明，涉及起始密码子的二级结构会阻止翻译起始。去除这个茎环会使BSt表达增加100倍。然而，表达水平仍然很低，仅占总细胞蛋白的0.5 - 1%。通过用trpLE密码子替换BSt cDNA的起始序列可以获得高水平表达。这些结果表明，除了二级结构外，BSt起始部分的核苷酸序列或氨基酸上下文与翻译起始步骤之一不相容。
