The influence of mRNA secondary structure on expression of endoglucanase inBacillus subtilis

Summary A gene for endoglucanase ofBacillus subtilis has been inserted into a Bacillus expression plasmid containing a strong BJ27 promoter and a synthetic ribosome binding site. Secondary structure analysis of mRNA showed the presence of a strong hairpin loop burying the SD sequence and the initiation codon. Alteration of secondary structure at this site by deletion analysis revealed a correlation between endoglucanase expression and accessibility of the ribosome binding site. Elimination of secondary structures increased endoglucanase expression over five-fold to a level at which endoglucanase occupied 60% of total protein which was secreted into culture medium.     

Effect of local secondary structure of mRNA on expression of recombinant human lymphotoxin inEscherichia coli

We substituted a few alternative codons close to the N-terminal coding region of human lymphotoxin (LT) gene for natural ones to avoid stable local secondary structures of mRNA, predicted in the 5-terminal 130 nucleotides of mRNA. With this modification, we have overexpressed a recombinant lymphotoxin (rLT) inEscherichia coli under the control of trc promoter. Most of the produced rLT was a soluble and active form.     

The influence of messenger RNA secondary structure on expression of an immunoglobulin heavy chain in Escherichia coli.

A gene for murine mu heavy chain immunoglobulin has been inserted into a bacterial expression plasmid containing the Escherichia coli trp promoter and ribosome binding site. A low level expression of mu protein was detected. Secondary structure analysis showed the presence of a hairpin loop burying the mu initiation codon. Alteration of secondary structure at this site by oligonucleotide replacement mutagenesis revealed a correlation between mu expression levels and accessibility of the ribosome binding site. Abolition of secondary structure increased mu protein expression over ninety-fold, to a level approximately equal to that of a trpE -mu fusion protein using the native trpE ribosome binding site.     

人防御素HNP-1基因在原核系统中的克隆与表达

通过化学合成法制备人防御素 HNP- 1基因 ,并将其构建于带有 GST基因的表达载体中 ,进行融合表达 ,表达融合蛋白可占菌体蛋白的 70 %左右 ,且主要以包含体形式存在。改变培养条件可适当提高可溶性融合蛋白的比例。融合蛋白通过凝胶色谱进行纯化 ,然后经凝血酶切释放出防御素多肽 ,并通过抑菌试验对其活性进行检测。结果显示 ,可能由于分子间的二硫键错配造成了防御素分子构象改变 ,使制备的防御素分子未显示出活性     

dam methylase from E. coli. Circular dichroism investigations of the secondary structure and influence of S-adenosylmethionine

The enzyme dam methylase which recognizes and methylates the adenine in the palindromic sequence GATC in DNA was isolated and the secondary structure was determined by CD spectroscopy and various predicting methods from the amino acid sequence. The interaction of dam methylase with S-adenosylmethionine was studied by CD spectroscopy indicating a decrease of the percentage of alpha-helix as the amount of S-adenosylmethionine bound to the enzyme was increased.     

Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli.

Escherichia coli expresses the beta and epsilon subunits of F1F0-ATP synthase at relative levels consistent with the 3:1 (beta/epsilon) stoichiometry in the holoenzyme. The mechanism of translational control of expression of the uncC gene (epsilon subunit) relative to the immediately 5' uncD gene (beta subunit) was examined. Previous expression studies and a computer analysis suggested the presence of an RNA secondary structure including the 3' end of uncD, the uncDC intergenic region, and the uncC Shine-Dalgarno sequence (S. D. Dunn and H. G. Dallmann, J. Bacteriol. 172:2782-2784, 1990). Analysis of in vitro-transcribed RNA by cleavage with RNases T1, V1, and CL3 and by chemical modification with dimethyl sulfate and diethyl pyrocarbonate confirmed a predicted structure. Introduction of premature uncD stop codons inserted 5' of the secondary structure strongly reduced epsilon expression, whereas stop codons inserted at positions within the secondary structure showed smaller effects, indicating that translational control of epsilon synthesis involves partial coupling to beta synthesis. Possible mechanisms by which the RNA secondary structure and the unfolding of this structure by translation of uncD may govern the level of uncC expression are discussed.     

The expression of human tumor necrosis factor in E. coli

cDNA of human natural TNF (n-TNF) obtained by stimulating human leukemic B cell line (Ball-1) with Sendai virus was cloned. Valine-started-TNF (V-TNF) gene was constructed from the cDNA and expressed in E.coli HB101 under the control of a trp promoter by the induction of 3-indoleacrylic acid. The expression level of V-TNF clone was about 10% of the total E.coli protein. On the other hand, the expression level of glutamine started-TNF (Q-TNF) gene having the same SD-ATG sequence which was constructed from V-TNF gene was as low as about 1/20 of that of V-TNF. The nucleotide sequence around ATG (-4 approximately +12) of Q-TNF gene was randomly changed without modifying the coded amino acid sequence, resulting to obtain high expression clones as similar TNF protein yield as that of V-TNF. These clones possessed A residue rich sequence around the initiation codon ATG. These results show that some correlation might exist between the high expression rate and A residue rich sequence around the initiation codon.     

argE基因在大肠杆菌中的表达优化

N-乙酰鸟氨酸脱酰基酶可在重组菌BL21（DE3）-pET22b-argE中表达。首先确定了该酶的细胞表达定位,再研究了诱导温度、诱导剂种类及浓度、诱导起始菌体密度、诱导时间等因素对重组菌生长及目的蛋白表达活性的影响。结果表明,IPTG和乳糖皆可诱导目的蛋白表达,乳糖的诱导效果优于IPTG。在诱导起始0D600为0．46时加入15g／L乳糖,20℃诱导18h最适于目的蛋白的活性表达。表达条件优化后,酶活从1．68U／mL提高至282．99U／mL,约为原来的168倍。     

mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli

Codon usage and thermodynamic optimization of the 5'-end of mRNA have been applied to improve the efficiency of human protein production in Escherichia coli. However, high level expression of human protein in E. coli is still a challenge that virtually depends upon each individual target genes. Using human interleukin 10 (huIL-10) and interferon alpha (huIFN-alpha) coding sequences, we systematically analyzed the influence of several major factors on expression of human protein in E. coli. The results from huIL-10 and reinforced by huIFN-alpha showed that exposing AUG initiator codon from base-paired structure within mRNA itself significantly improved the translation of target protein, which resulted in a 10-fold higher protein expression than the wild-type genes. It was also noted that translation process was not affected by the retained short-range stem-loop structure at Shine-Dalgarno (SD) sequences. On the other hand, codon-optimized constructs of huIL-10 showed unimproved levels of protein expression, on the contrary, led to a remarkable RNA degradation. Our study demonstrates that exposure of AUG initiator codon from long-range intra-strand secondary structure at 5'-end of mRNA may be used as a general strategy for human protein production in E. coli.     

Mx基因稀有密码子和mRNA结构及大肠杆菌表达 优化

通过对稀有密码子和mRNA翻译起始区二级结构的分析, 构建了4种重组表达菌株BL21(DE3)/pET-Mx, Rosseta(DE3)/pET-Mx, BL21(DE3)/pGEX-Mx和Rosseta(DE3)/pGEX-Mx, 在大肠杆菌中进行Mx基因的表达, Rosseta(DE3)/pET-Mx和Rosseta(DE3)/pGEX-Mx重组表达菌中都获得了表达, Western blotting检测到了特异的75 kDa表达产物。实验结果证明稀有密码子和mRNA翻译起始区二级结构对Mx 蛋白表达都有影响, 选择适用于稀有密码子表达的菌株Rosetta(DE3)有利于Mx蛋白的表达, 同时翻译起始区二级结构能值较低的表达载体pGEX-Mx获得的表达量明显增高。实验中首次获得了重组表达鸡全长Mx蛋白的大肠杆菌重组菌。     

Two-cistronic expression plasmids for high-level gene expression in Escherichia coli preventing translational initiation inhibition caused by the intramolecular local secondary structure of mRNA

Two-cistronic expression plasmids for the wild-type solubilized domain of porcine NADPH-cytochrome P450 reductase (PsCPR) gene in Escherichia coli were systematically constructed using a solubilized domain of porcine cytochrome b5 gene (Psb5 gene) or a derivative of it as the first cistron to examine their utility for second gene expression preventing the translational inhibition caused by the intramolecular local secondary structure of mRNA at the ribosome-binding site (RBS). The mRNAs from the plasmids lacking an RBS for the second cistron (SD2) accumulated very low levels of PsCPR, while those from the plasmids having an SD2 accumulated higher levels of PsCPR. The level of accumulation of PsCPR by the mRNA from plasmid pCbSD-T-CPR-3, which has an SD2 upstream of the termination codon of the first cistron, was higher than for those with an SD2 in the intercistronic region. The predicted intramolecular local secondary structures at the SD2 of mRNAs from these plasmids were stable enough to cause translational initiation inhibition. These results indicate that the use of a two-cistronic expression plasmid is an effective way to overcome translational initiation inhibition. Improved plasmids, pCP1 and pCP2P, were constructed from pCbSD-T-CPR-3. Using these plasmids, the solubilized donain of porcine NADH-cytochrome b5 reductase was also highly accumulated on prevention of the translational initiation inhibition. These plasmids are expected to be useful tools for the comprehensive high-level expression of heterologous genes in E. coli cells.     

Cloning a synthetic gene for human stefin B and its expression in E. coli

A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector. The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E. coli as a fusion protein with beta-galactosidase and as a native protein. The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.