中性蛋白酶基因诱导型表达分泌载体的构建

利用PCR方法分别扩增出sacB基因的启动子-信号肽序列（sacR）和枯草芽孢杆菌中性蛋白酶的前肽-成熟肽序列,将两者连接后克隆入载体pHP13中,构建了含有中性蛋白酶基因的诱导型表达分泌载体pHP13SN,再将其转化入枯草杆菌DB104,获得基因工程菌DB104(pHP13SN)。中性蛋白酶基因在蔗糖的诱导和sacR的调控下实现了分泌表达,并获得了具有生物学活性的中性蛋白酶。     

Use of alkaline phosphatase fusions to study protein secretion in Bacillus subtilis.

We have constructed a vector designed to facilitate the study of protein secretion in Bacillus subtilis. This vector is based on a translational fusion between the expression elements and signal sequence of Bacillus amyloliquefaciens alkaline protease and the mature coding sequence for Escherichia coli alkaline phosphatase (phoA). We show that export of alkaline phosphatase from B. subtilis depends on a functional signal sequence and that alkaline phosphatase activity depends upon secretion. The vector design facilitates the insertion of heterologous coding sequences between the signal and phoA to generate three-part translational fusions. Such phoA fusions are easily analyzed by monitoring alkaline phosphatase activity on agar plates or in culture supernatants or by immunological detection. Exploitation of this methodology, which has proven to be extremely useful in the study of protein secretion in E. coli, has a variety of applications for studying protein secretion in B. subtilis.     

枯草杆菌碱性蛋白酶基因诱导表达载体的构建

以PCR方法扩增sacB基因的启动子-信号肽序列(称为sacR),将其与枯草芽孢杆菌碱性蛋白酶的前肽-成熟酶基因连接后克隆入载体pUBH,构建了含碱性蛋白酶基因的分泌型诱导表达载体pUBS,将其转化枯草芽孢杆菌DB403后,获得基因工程菌DB403(pUBS)。碱性蛋白酶基因在sacR的调控和蔗糖的诱导下实现了表达分泌,获得了具生物学活性的碱性蛋白酶。     

Far different levels of gene expression provided by an oriented cloning system in Bacillus subtilis and Escherichia coli

A gene expression system for both Bacillus subtilis and Escherichia coli was developed. The expression vector, pHASH102, produces any combination of promoter and open reading frame to be expressed based on the T-extended cloning method. Because the pHASH series vectors are designed to shuttle between the genome and a high copy plasmid in B. subtilis, the expression profiles of copy number dependence can be examined systematically. We demonstrated that vectors with Pr, Pspac, and PS10 promoters are suitable for the overexpression of GFPuv. Moreover, aadK encoding aminoglycoside 6-adenylyltransferase (a streptomycin-resistance gene) of B. subtilis was successfully overexpressed in both B. subtilis and E. coli. These highly expressed GFPuv and aadK genes can be used as a genetic marker for both organisms.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease.

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His₆-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.     

High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Prochymosin expression in Bacillus subtilis

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.     

Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome.

Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.     

Protein secretion in Bacillus subtilis as influenced by the combination of signal sequence and the following mature portion

Abstract We have constructed secretion vector plasmids that have the signal sequence of the Bacillus licheniformis penicillinase gene ( penP ) or the Bacillus stearothermophilus α-amylase gene ( amyT ). We have also constructed penP, amyT and hsa (human salivary α-amylase gene) cartridges. Each of these cartridges was cloned on secretion vectors in Bacillus subtilis , and enzyme production was examined. When amyT vector was used, nearly the same efficiency of enzyme secretion was observed for amyT and penP cartridges. When penP vector was used, enzyme secretion for amyT decreased to about 3% of that for penP cartridges. The eukaryotic gene hsa was hardly expressed in any secretion vectors in B. subtilis .     

A new family of sugar-inducible expression vectors for Escherichia coli.

A set of 11 expression vectors was constructed, each of them harbouring a cloning cassette under the control of the araB promoter. Some of these vectors enable expression of foreign proteins in the cytoplasm, while others include a synthetic sequence coding for a very efficient secretion signal sequence. Other features are an f1 origin of replication (in plus or minus orientation) and a promoter(up) mutation that enhances the already very high level of expression from these vectors. With such a versatile vector family, cloning, sequencing and site-directed mutagenesis can be performed on the same vector, and the level of expression can be defined according to the specific constraints of a given protein.     

An Improved pPZP Vector for Agrobacterium-mediated Plant Transformation

We report a new and improved pPZP vector (pPZP3425) for efficient plant transformation. This vector is derived from the widely used pPZP100 series of binary Agrobacterium vectors. One disadvantage of these vectors is the use of chloramphenicol resistance for selection in Escherichia coli and Agrobacteria. We have therefore included a kanamycin resistance gene for selection in Agrobacterium. Furthermore, the strong 35S CaMV promoter driving the plant resistance gene has been replaced by the weaker nos promoter because it has been shown that the 35S promoter driving the plant resistance marker can lead to ectopic expression of the transgene. During replacement of the 35S promoter, the NcoI site within the plant resistance gene has been removed, and NcoI can now be used for cloning purposes within the expression cassette which consists of an intron-containing gus gene driven by a strong constitutive promoter (35S promoter with doubled enhancer plus omega-element as translational enhancer). Thus, a single vector can conveniently be used for two purposes: (1) for overexpression of proteins by replacing the gus gene by the coding sequence of choice and (2) for creation of promoter:gus fusions by substituting the constitutive promoter by any other promoter. We demonstrate the usefulness of this vector for cloning a promoter:gus fusion and in planta transformation of Arabidopsis.     

生长激素信号肽可诱导重组蛋白外分泌表达

重组蛋白质的表达是生物医药开发、基因功能和作用机理研究中关键技术环节.虽然细菌表达体系由于表达量大、经济等而被广泛采用,但由于其不能提供许多蛋白质必需的翻译后修饰如糖基化等,所表达的蛋白又多以不可溶包涵体形式存在,变性复性过程复杂,产率低,因此真核细胞表达体系如CHO、COS等成为活性要求高的蛋白质表达的首选[1].     

Cloning and Transfer of the Salmonella Pathogenicity Island 2 Type III Secretion System for Studies of a Range of Gram-Negative Genera

The engineering of bacterial strains with specific phenotypes frequently requires the use of blocks or “cassettes” of genes that act together to perform a desired function. The potential benefits of utilizing type III secretion systems in this regard are becoming increasingly realized since these systems can be used to direct interactions with host cells for beneficial purposes such as vaccine development, anticancer therapies, and targeted protein delivery. However, convenient methods to clone and transfer type III secretion systems for studies of a range of different types of bacteria are lacking. In addition to functional applications, such methods would also reveal important information about the evolution of a given type III secretion system, such as its ability to be expressed and functional outside of the strain of origin. We describe here the cloning of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system onto a vector that can be easily transferred to a range of gram-negative bacterial genera. We found that expression of the cloned SPI-2 system in different Gammaproteobacteria and Alphaproteobacteria (as monitored by SseB protein levels) is dependent on the bacterial strain and growth medium. We also demonstrate that the cloned system is functional for secretion, can direct interactions with macrophages, and can be used as a novel tool to analyze the predicted interaction of SseB with host cells. This work provides a foundation for future applications where the cloned SPI-2 region (or other cloned type III systems) can provide a desired function to an engineered gram-negative strain.     

Effect of in vitro DNA rearrangement in the NH2-terminal region of the penicillinase gene from Bacillus licheniformis on the mode of expression in Bacillus subtilis

We have constructed secretion vector plasmids that have unique BglII sites within or near the signal sequence of Bacillus licheniformis penicillinase, and have also constructed penicillinase cartridges that lack either one, two or three of the processing sites for the membrane-bound, exo-large and exo-small enzymes. Each of these penicillinase cartridges was cloned on secretion vectors in Bacillus subtilis, and enzyme production was examined. The presence of both the signal sequence and the three host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B. subtilis. The presence of any of the processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium. When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the three processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge. Molecular mass determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme.     

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis.

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.