Gene cloning and expression in lactic streptococci

Abstract Recent developments have made the mesophilic lactic streptococci, widely used in dairy fermentations, accessible to genetic manipulation. Several host-vector systems have been described which currently are used in the cloning and expression of homologous and heterologous genes. The essential elements of these systems, the various cloning strategies and the first successful cloning experiments are described with emphasis on the molecular organization of proteinase genes. In addition, the organization and nucleotide sequence of signals which are involved in gene expression in lactic streptococci are summarized.     

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.     

大肠杆菌高效表达重组蛋白策略

大肠杆菌表达系统是基因表达技术中发展最早和目前应用最广的经典表达系统。利用该系统表达重组蛋白具有许多优越性,但其表达效率受诸多因素的影响。本文综述国内外利用大肠杆菌表达系统高效表达外源蛋白的策略,主要包括选择合适的启动子、改变信号肽结构、提高mRNA稳定性、提高翻译效率、表达稀有密码子、降低包涵体形成及蛋白降解,利用融合蛋白与分子伴侣、调控发酵条件实现高密度培养等。     

Enzyme Free Cloning for high throughput gene cloning and expression

Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning (LIC). Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC (79%). EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.     

烟实夜蛾性信息素合成激活肽基因的分子克隆

根据家蚕Bombyx mori和玉米夜蛾Helicoverpa zea的性信息素合成激活肽基因序列,设计若干套引物, 以烟实夜蛾Heliothis assulta基因组DNA为模板进行PCR扩增, 得到0.63 kb的特异性DNA片段。该片段克隆进适当载体,序列测定和同源比较, 查明烟实夜蛾的基因组中存在性信息素合成激活肽基因。烟实夜蛾的性信息素合成激活肽由33个氨基酸组成, C末端是FXPRL结构,是目前发现的第4种昆虫性信息素合成激活肽。在该神经肽第14和第15个氨基酸之间, 插入一个0.42 kb的内含子。 进一步的分析证明了烟实夜蛾的性信息素合成激活肽基因在潜成虫期的食道下神经节中表达。     

Molecular cloning and sequence of theBacillus stearothermophilus translational initiation factor IF2 gene

Summary The structural gene for theBacillus stearothermophilus initiation factor IF2 was localized to a 6 kbHindIII restriction fragment by cross-hybridization with theSstI-SmaI fragment of theEscherichia coli infB gene. This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous inE. coli IF2, EF-Tu, EF-G and the humanras1 oncogene protein. After cloning into pACYC177, theHindIII fragment was further analysed by restriction mapping and cross-hybridization. A smaller (2.2 kb)SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method. This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus. This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlappingClaI-SstI fragment which was also subcloned and sequenced. Overall, theB. stearothermophilus IF2 gene codes for a protein of 742 amino acids (M_r=82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the correspondingE. coli IF2 molecule. When cloned into an expression vector under the control of the λP_L promoter, theB. stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels inE. coli cells.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Expression of Actinobacillus pleuropneumonia gene coding for Apx I protein in Escherichia coli

This study presents cloning and expression of Actinobacillus pleuropneumoniae Apx I toxin in Escherichia coli expression system to produce fusion protein for the subsequent immunological studies. The gene coding Apx I toxin was amplified from the A. pleuropneumoniae serotype 10 DNA using polymerase chain reaction and cloned to vector under the control of strong, inducible T7 promoter. The presence of insert was confirmed by PCR screening and sequencing after the propagation of recombinant DNA in E. coli cells. The gene coding A. pleuropneumoniae Apx I toxin was extended with a segment to encode a polyhistidine tag linked to its C-terminal sequence allowing a one-step affinity purification of the complex with Ni-NTA resin. Expression of the Apx I coding sequence in E. coli resulted in the formation of insoluble inclusion bodies purified according to a standard purification protocol. The ease of this expression system, the powerful single-step purification and low costs make it possible to produce Apx I in large amounts to further study the role of Apx I in physiological processes.     

南方鲇Vasa基因两种亚型cDNA的克隆及其表达

采用RT-PCR和RACE相结合的方法,从南方鲇分离到Vasa基因的两个亚型scVasa和scVaga-s。它们是同一基因在5′端经选择性剪接的产物,其cDNA全长分别为2525bp和2438bp,编码662和641个氨基酸。两者均具有DEAD-box家族成员特有的8个保守基序和Vasa的典型特征。南方鲇Vasa与银鲫相似性最高（73．3％）。两个亚型均特异地表达于雌雄性腺中。原位杂交结果表明：scVasa主要在卵巢Ⅰ、Ⅱ时相的卵母细胞和精巢的精原细胞和初级精母细胞中表达。半定量PCR结果显示,在生殖周期中,两种亚型在以Ⅱ时相卵母细胞为主体的卵巢恢复期表达均高于以Ⅲ-Ⅳ时相卵母细胞为主体的卵黄生成期[动物学报54（6）：1051—1060,2008]。     

Molecular cloning of the cDNA coding for regucalcin and its mRNA expression in mouse liver: The expression is stimulated by calcium administration

The molecular cloning of the cDNA coding for a Ca2+-binding proteinregucalcin and its mRNA expression in mouse liver were investigated. ThecDNA clone encoding a regucalcin was isolated from a mouse liver cDNAlibrary and sequenced. Analysis of the sequence of the cloned cDNA showedthat the cDNA encoded the complete amino acid sequence of the mouseregucalcin molecule; the cDNA had an open reading frame of 897 bp. Mouseregucalcin was composed of 299 amino acid residues, and its molecular weightwas estimated to be 33,406 Da. The amino acid sequence of mouse regucalcinhad 94% homology, as compared with that of rat regucalcin. Northernblot analysis with the mouse liver cDNA probe revealed that mouse regucalcinmRNA was mainly present in the liver but only slightly in the kidney with asize of 1.8 kb. Hepatic regucalcin mRNA level of male mouse was higher thanthat of female mouse. A single intraperitoneal administration of calciumchloride (5, 15, and 30 mg Ca2+/100 g body weight) to mice induced aremarkable increase in regucalcin mRNA in the liver; the increase inregucalcin mRNA levels at 30 min after calcium administration wasdose-dependent. The present results demonstrate that regucalcin mRNA in miceis uniquely expressed in the liver, and that its expression is stimulated bycalcium administration.     

Molecular cloning of a possible excretion protein of Vibrio cholerae

Abstract The gene for a Vibrio cholerae protein of about kDa (kilodalton) has been cloned and its location within the 1.9-kb cloned DNA fragment determined by transposon insertion and deletion analyses. The proteins encoded within the various plasmids have been analyzed in Escherichia coli K-12 minicells. The 25-kDa protein when expressed in E. coli K-12 allows the release of the periplasmic deoxyribonuclease. It is a minor protein suggesting that the release of DNase is not an artefact due to membrane damage. It is possible that this protein functions as part of an excretion system.
Results with transposon Tn 1725 insertions suggest that it contains a termination site in one orientation and a promoter in the other.     

Molecular cloning of the S-layer protein gene of Campylobacter rectus ATCC 33238

Abstract A genomic DNA library of Campylobacter rectus (Wolinella recta) ATCC 33238 was constructed using bacteriophage lambda EMBL3 as a vector. One clone expressing the S-layer protein was identified immunologically with the antiserum to the S-layer protein of C. rectus ATCC 33238. Western immunoblotting using monoclonal antibodies directed against the S-layer protein showed a single blot of recombinant protein at 150 kDa, suggesting that this clone contained the entire coding region of the S-layer protein gene. Additionally, the S-layer protein gene was subcloned into plasmid vector pUC18. Southern hybridization revealed that the S-layer protein gene was present on the chromosome of C. rectus as a single-copy gene, and that there were minor heterogeneities among the S-layer protein genes of clinical isolates. Moreover, a spontaneous stable mutant strain, which has no S-layer expression, also conserved the full-length gene of the S-layer protein.     

White and green screening with circular polymerase extension cloning for easy and reliable cloning

Cloning is an essential prerequisite to test protein design and engineering ideas. However, it is often time consuming, unreliable, and therefore frustrating. Here, we present a streamlined cloning strategy that incorporates a powerful white and green screening protocol to identify colonies with inserts. We use circular polymerase extension cloning, which is both ligation and sequence independent. Furthermore, our entire procedure requires only three quick steps and one enzyme making it easy to use, inexpensive, and tractable. We anticipate that this method will be particularly useful for protein engineers who frequently subclone or make focused deletion, insertion, or substitution libraries.     

马链球菌透明质酸合成酶基因的分子克隆及表达

根据Streptococcus equisimilis、Streptococcus pyogenes、Streptococcus uberis三种链球菌透明质酸合成酶(seHAS、spHAS、suHAS)基因的高度保守区,设计一对简并引物,用两次PCR从Streptococcus equi的总DNA中扩增出sqHAS基因。构建表达质粒pSE-sqHAS并转化大肠杆菌DH5α,诱导培养后在细胞膜中检测到sqHAS蛋白及活性。利用携带该酶的细胞膜以UDP-GlcA和UDP—GlcNAc为底物在体外合成了分子量为3.6×10~6Da的HA,分别是发酵法生产和提取法生产的HA的分子量的2.5倍和5倍左右。马链球菌透明质酸合成酶基因的克隆及表达,国内外文献尚未见报道。本研究为体外酶法生产透明质酸做了初步探索。     

棉花粉蚧化学感受蛋白基因PsCSP10的克隆及表达谱分析

本研究克隆出了棉花粉蚧Phenacoccus solenopsis Tinsley化学感受蛋白(CSPs)基因Ps CSP10的全长c DNA(Gen Bank登录号:KT958555),其核苷酸序列长640 bp,编码128个氨基酸,预测其成熟蛋白分子量14.99 k D,等电点6.61,且含有4个保守的半胱氨酸,符合昆虫CSPs的典型特征。该基因编码的氨基酸序列和其他昆虫化学感受蛋白基因编码的氨基酸序列相似性为50%-56%。应用Real-time PCR测定的结果表明棉花粉蚧各发育阶段中Ps CSP10均有表达,但在雄成虫中的相对表达量显著高于其他发育阶段。在分别用扶桑Hibiscus rosa-sinensis L.、棉花Gossypium hirsutum L.、马缨丹Lantana camara L.饲养获得的雄成虫中Ps CSP10相对表达量不存在差异。研究结果为进一步明确棉花粉蚧中Ps CSP10的功能奠定了基础。     

Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli

We describe here the construction of a 10-Gateway-based vector set applicable for high-throughput cloning and for expressing recombinant proteins in Escherichia coli. Plasmids bear elements required to produce recombinant proteins under control of the T7 promoter and encode different N-terminal partners. Since the vector set is derived from a unique backbone, a consistent comparison of the impact of fusion partner(s) on protein expression and solubility is easily amenable. Finally, a sequence encoding a six-histidine tag has been inserted to be in frame with the cloned open reading frame either at its C terminus or at the N terminus, giving the flexibility of choosing the six-histidine tag location for further purification. To test the applicability of our vector set, expression and solubility profile and six-histidine tag accessibility have been demonstrated for two Bacillus subtilis signaling proteins' encoding genes (SBGP codes E0508 and E0511).