High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system

The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.     

The stability of pET21 + PA,a pET derived plasmid,under different culture conditions

The recombinant plasmid pET21 + PA that has been deposited at Genbank with accession number EF550209 was constructed by inserting the 1,700-bp PA (protective antigen of Bacillus anthracis) recombinant gene into Xho I/Hind Ш sites of the pET21b + vector under the control of the T7 promoter for highly expressing PA. pET21 + PA was cloned into Escherichia coli BL21 strain. The high activity of T7 RNA polymerase could make a powerful expression system for high-level expression of the recombinant proteins. However, during the large-scale production of recombinant proteins, the productivity of a fermentation process is directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and target protein yield including antibiotic concentrations, the time of induction by IPTG, and the number of successive cultures. The results indicated that the plasmid pET21 + PA is completely stable after the fiftieth generation. Loss of plasmid and structural change were not detected but the yield of protein production was decreased by about 10% in generation 50. These data would be useful for the industrial production of the recombinant PA vaccine and other recombinant proteins.     

Development and application of an arabinose-inducible expression system by facilitating inducer uptake in Corynebacterium glutamicum

Corynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species use lac-derived promoters from Escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains the L-arabinose regulator AraC, the P(BAD) promoter from the araBAD operon, and the L-arabinose transporter AraE, all of which are derived from E. coli. The level of inducible P(BAD)-based expression could be modulated over a wide concentration range from 0.001 to 0.4% L-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control of P(BAD) promoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case in E. coli, P(BAD) induction was not significantly affected in the presence of different carbon sources in C. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of the odhI gene from C. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering of C. glutamicum.     

A freeze-thaw method for disintegration of Escherichia coli cells producing T7 lysozyme used in pBAD expression systems

The pLysN plasmid containing the T7 lysozyme gene under control of the lac promoter was constructed to facilitate cell disintegration after expression of recombinant proteins in arabinose-induced expression systems. The usefulness of this plasmid was tested in Escherichia coli TOP10 and E. coli LMG194 cells carrying pBADMHADgeSSB plasmid containing Deinococcus geothermalis SSB protein gene under control of the araBAD promoter. The results showed that low-level expression of T7 lysozyme did not interfere with the target SSB protein production, and that the freezing-thawing treatment was sufficient for disruption of the E. coli cells producing low amounts of T7 lysozyme.     

通过一种新方法使T7基因Ⅰ置换araBAD基因簇

目的：用T7RNA聚合酶基因T7基因Ⅰ置换大肠杆菌基因组中的araBAD基因簇。方法：通过分析大肠杆菌基因组中araBAD基因序列,设计两侧同源臂,构建高拷贝数的打靶质粒,并且在这个质粒上的打靶片段两侧各加上一个归巢内切酶位点;将辅助质粒和打靶质粒同时转化到宿主体内,在L-阿拉伯糖的诱导下表达归巢内切酶和Red重组酶,实现体内重组,使T7基因I置换araBAD基因簇。结果：在4株不同的大肠杆菌中用T7基因I置换了araBAD基因簇。结论：成功地将T7基因Ⅰ整合到araBAD基因位置,为构建PAra-PT7表达系统奠定了基础。     

Expression of E. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae

The Escherichia coli araBAD operon consists of three genes encoding three enzymes that convert L-arabinose to D-xylulose-5 phosphate. In this paper we report that the genes of the E. coli araBAD operon have been expressed in Saccharomyces cerevisiae using strong promoters from genes encoding S. cerevisiae glycolytic enzymes (pyruvate kinase, phosphoglucose isomerase, and phosphoglycerol kinase). The expression of these cloned genes in yeast was demonstrated by the presence of the active enzymes encoded by these cloned genes and by the presence of the corresponding mRNAs in the new host. The level of expression of L-ribulokinase (araB) and L-ribulose-5-phosphate 4-epimerase (araD) in S. cerevisiae was relatively high, with greater than 70% of the activity of the enzymes in wild type E. coli. On the other hand, the expression of L-arabinose isomerase (araA) reached only 10% of the activity of the same enzyme in wild type E. coli. Nevertheless, S. cerevisiae, bearing the cloned L-arabinose isomerase gene, converted L-arabinose to detectable levels of L-ribulose during fermentation. However, S. cerevisiae bearing all three genes (araA, araB, and araD) was not able to produce detectable amount of ethanol from L-arabinose. We speculate that factors such as pH, temperature, and competitive inhibition could reduce the activity of these enzymes to a lower level during fermentation compared to their activity measured in vitro. Thus, the ethanol produced from L-arabinose by recombinant yeast containing the expressed BAD genes is most likely totally consumed by the cell to maintain viability.     

枯草芽孢杆菌C-36内切葡聚糖酶基因的克隆及其在大肠杆菌中融合表达

以自行分离筛选出的天然枯草芽孢杆菌（Bacillus subtilis）C-36的染色体DNA为模板,PCR扩增得到含有内切葡聚糖酶基因的DNA片段,将其克隆到pMD-18T载体中,序列分析表明,克隆得到的DNA片段全长1602bp,编码一个含有499个氨基酸的多肽。与其他芽孢杆菌内切葡聚糖酶基因序列比对,其核苷酸同源率为90％～93％,其编码的氨基酸序列的同源性在90％～98％,已将此基因注册GenBank（DQ782954）。将含内切葡聚糖酶基因的重组克隆质粒进行亚克隆,用Kpn I和EcoR I双酶切后,与相同酶切的表达载体pET-32a相连接,并导入大肠杆菌BL21中表达。蛋白质电泳实验结果表明在6．47×10^4处有表达蛋白带。经测定表达蛋白比酶活力达99．02U／mL,为出发菌C-36（63．78U／mL）的1．55倍。     

Development of a fed-batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat

To effectively achieve tight regulation and high-level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30 degrees -->41 degrees C stepwise upshift, the shake-flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30 degrees C. By exploiting the fed-batch fermentation approach, the recombinant cell batch initially kept at 30 degrees C in a lab-scale fermentor was exposed to 41 degrees C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37 degrees C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880-fold over that obtained at the shake-flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale-up of recombinant proteins.     

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.     

精氨酸脱亚胺酶在大肠杆菌中的分泌型表达

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arc A克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢB中,经鉴定得到重组质粒pBAD-ADI。将重组质粒转化大肠杆菌TOP10F'后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002%(w/v),25℃下诱导5 h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同)。采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL。SDS-PAGE分析显示,重组蛋白大小约为46 kD。     

Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)

Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.     

人内皮生长抑制素基因克隆表达及其抑瘤作用

内皮生长抑制素（ｅｎｄｏｓｔａｔｉｎ是最近报道具有抑制肿瘤生长和铁蛋白质,为此,从正常人肝细胞株Ｌ０２中抽提总ＲＮＡ,以ＲＴ－ＰＣＲ法获得了人内皮生长抑制素编码序列,序列分析表明,内皮生长抑制素ＣＤＮＡ开放阅读框架全长５５５ｂｐ,此基因（ＧｅｎＢａｎｋ登录号为Ａ地８４０６０）,共编码１、８４个氨基酸残序列为５个碱基与文献报道不同,蛋白质序列中有３个氨基酸残基与文献报道不同,说明存在人种间差异。将人     

High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei

The cellobiase gene from Aspergillus niger was cloned and connected with the strong promoter Pcbh1 from Trichoderma reesei to construct a recombinant plasmid pHB9 with the hygromycin B resistance marker. The plasmid was transformed into conidia of T. reesei using the modified PEG-CaCl₂ method. Main factors effecting the transformation were discussed and about 99-113 transformants/μg DNA could be obtained under optimal conditions. It was found that the molecular mass of the recombinant cellobiase was about 120 kDa by SDS-PAGE analysis. The activity of cellobiase could reach 5.3 IU/ml after 48 h fermentation, which was as high as 106 times compared with that of the host strain. Meanwhile, the filter paper activity of recombinant T. reesei was 1.44-fold of the host strain. Saccharification of corncob residue with the crude enzyme showed that the hydrolysis yield (84.2%) of recombinant T. reesei was 21% higher than that (69.5%) of the host strain.     

基因重组大肠杆菌表达血红素加氧酶-1及培养条件优化

【背景】血红素加氧酶-1 (HO-1)具有抗氧化应激、抗凋亡和抗纤维化等多种生理效应,有望成为一种新型药物应用于临床疾病的治疗。【目的】构建表达HO-1的基因重组大肠杆菌(Escherichiacoli),并优化其表达培养条件,实现HO-1高产率的表达。【方法】PCR法克隆集胞藻(Synechocystissp.)PCC6803的HO-1基因(ho1),构建重组质粒pET-28a-ho1,转化大肠杆菌BL21(DE3)菌株,单因素实验优化表达培养基的种类、诱导剂添加时间、诱导培养时间、诱导剂浓度和诱导培养温度。【结果】构建了表达HO-1的基因重组大肠杆菌BL21(DE3)/pET-28a-ho1菌株,用甘油(GY)培养基培养至菌体浓度OD_(600)约为0.8时,加入终浓度为0.1 mmol/L的IPTG诱导,30°C诱导培养6 h,HO-1的表达量最高,Ni-NTA柱分离纯化得到的HO-1收率占细胞总蛋白的10.9%。【结论】获得了可溶性表达HO-1的基因重组大肠杆菌及其较佳的培养条件,为进一步研究集胞藻来源的HO-1的酶学性质和应用奠定了基础。     

霍乱毒素B亚单位基因（CtxB）的克隆及其表达

从霍乱弧菌中抽提基因组ＤＮＡ,用ＰＣＥＲ方法获取霍乱毒素Ｂ亚单位基因（ＣｔｘＢ）。序列分析结果表明,ＣｔｘＢ基因编码１２４个氨基酸,其中编码６２位Ｔｈｒ的密码子与文献报道有差异。将ＣｔｘＢ基因插入质粒ｐＧＥＸ－４Ｔ－２,构建ｐＧＥＸ－ＣＴＸＢ表达质粒,转化大肠相菌ＢＬ２１（ＤＥ３０,筛选表达菌株ＣＴＸＢ／ＢＬ２１。工程株经ＩＰＴＧ诱导表达,可产生大量的表达蛋白,经ＳＤＳ－ＰＡＧＥ分析,融合蛋白分子     

Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nuclease

Expression strains of Escherichia coli BL21(DE3) overproducing the E. coli m(5)C McrA restriction protein were produced by cloning the mcrA coding sequence behind a T7 promoter. The recombinant mcrA minus BL21(DE3) host produces active McrA as evidenced by its acquired ability to selectively restrict the growth of T7 phage containing DNA methylated in vitro by HpaII methylase. The mcrA coding region contains several non-optimal E. coli triplets. Addition of the pACYC-RIL tRNA encoding plasmid to the BL21(DE3) host increased the yield of recombinant McrA (rMcrA) upon induction about 5- to 10-fold. McrA protein expressed at 37 degrees C is insoluble but a significant fraction is recovered as soluble protein after autoinduction at 20 degrees C. rMcrA protein, which is predicted to contain a Cys(4)-Zn(2+) finger and a catalytically important histidine triad in its putative nuclease domain, binds to several metal chelate resins without addition of a poly-histidine affinity tag. This feature was used to develop an efficient protocol for the rapid purification of nearly homogeneous rMcrA. The native protein is a dimer with a high alpha-helical content as measured by circular dichroism analysis. Under all conditions tested purified rMcrA does not have measurable nuclease activity on HpaII methylated (Cm(5)CGG) DNA, although the purified protein does specifically bind HpaII methylated DNA. These results have implications for understanding the in vivo activity of McrA in "restricting" m(5)C-containing DNA and suggest that rMcrA may have utility as a reagent for affinity purification of DNA fragments containing m(5)C residues.     

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.     

Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain

AIMS: Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. METHODS AND RESULTS: The keratinase gene with and without leader sequence from the chromosomal DNA of Bacillus licheniformis MKU3 was amplified by PCR and cloned into pET30b and transferred into Escherichia coli BL21. The ker gene without leader sequence only expressed in E. coli and the recombinant strain produced an intracellular keratinase activity of 74.3 U ml(-1). The ker gene was further subcloned into E. coli-Bacillus shuttle vector, pWH1520. Bacillus megaterium ATCC 14945 carrying the recombinant plasmid pWHK3 expressed the ker gene placed under xylA promoter and produced an extracellular keratinase activity of 95 U ml(-1). Response surface methodology (RSM) was employed to optimize the fermentation condition and to improve the level of keratinase production by the recombinant strain. A maximum keratinolytic activity of 166.2 U ml(-1) (specific activity, 33.25 U mg(-1)) was obtained in 18 h of the fermentation carried out with an initial inoculum of 0.4 OD600 nm and xylose concentration of 0.75% w/v. CONCLUSIONS: Bacillus licheniformis keratinase was cloned and successfully expressed using T7 promoter in E. coli and xylose inducible expression system in B. megaterium. Response surface methodology was employed to optimize the process parameters, which resulted in a three-fold higher level of keratinase production by the recombinant B. megaterium (pWHK3) than the wild type strain B. licheniformis MKU3. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. megaterium is a suitable host for the expression of cloned genes from heterologous origin. Optimization of fermentation conditions improved the keratinase production by B. megaterium (pWHK3) and suggested that this recombinant strain could be used for the production of keratinase.