Identification of the plasmid-encoded immunity protein for colicin E1 in the inner membrane of Escherichia coli

A set of plasmids containing portions of the Col El plasmid were transformed into recA⁻ cells. These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins. UV-irradiated cells label a 14.5 kDa band if they are phenotypically immune to colicin E1, and do not contain this band if they are sensitive to colicin E1. We conclude that the 14.5 kDa protein is the colicin E1 immunity protein. When the inner and outer membranes of these cells are fractionated, the labelled band appears in the inner membrane. The immunity protein must be an intrinsic inner membrane protein, confirming the predictions made by hydrophobicity calculations from primary sequence data.

Maxicell Col El plasmid Immunity protein Hydrophobicity calculation     

Demonstration of alternative splicing of a pre-mRNA expressed in the blood stage form of Plasmodium falciparum.

By screening of a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum raised against a 41-kDa protein band, which was shown to confer protective immunity to monkeys, the phage clone 41-3 was identified. The entire 41-3 gene was isolated, and its coding regions were determined by amplification and sequencing of 41-3 specific mRNA fragments. The 41-3 gene has a complex structure consisting of nine exons, encoding 375 amino acids in total with a calculated molecular weight of 43,400. Provided that the N-terminal hydrophobic residues function as signal sequence which is cleaved off, the molecular weight of the 41-3 protein decreases to 41,200 and could therefore be considered to be a component of the protective Mr = 41,000 protein band. Indeed, a 41-kDa protein could be detected by Western blot analysis using antisera raised against different recombinant expression products of the 41-3 gene. We furthermore demonstrate an alternative splice process for the mRNA precursor transcribed from the 41-3 gene to yield at least three distinct mRNAs. The major splice product carries all exons E1 to E9, whereas at least two minor 41-3 mRNA species can be identified which show deletions in the region between exons E5 and E7. The possible role of this differential splice process for the parasite is discussed.     

Assignment of the gene for the small nuclear ribonucleoprotein E (SNRPE) to human chromosome 1q25-q43

Small nuclear ribonucleoproteins (snRNPs), which are composed of various U RNAs and several proteins, are components of the mRNA splicing apparatus. The snRNP protein E is encoded by a multigene family which consists of a single expressed gene and several processed pseudogenes. We have used somatic cell hybridization, in situ hybridization, and linkage analysis to both physically and genetically map the expressed E protein gene to human chromosome 1q25-43, with the most probable location being band 1q32. In addition to the snRNP E protein gene, two other snRNP components--the U1 RNA true multigene family and a group of class I U1 pseudogenes--are located on human chromosome 1.     

人源抗TNF—α抗体Fab基因的单链化及其表达和纯化

构建人源抗TNF－α单链抗体基因,并尝试其在E.coli中的表达和纯化,采用人工接头,按VH－linkerVL的结构将人源抗TNF－a的VH,VL基因拼接成单链抗体（ScFv)基因,并将构建好的ScFv基因插入表达载体pQE30,转染E.coli M15,以IPTG诱导表达,Ni-NTA树脂纯化表达产物,构建的ScFv基因长723bp, 列分析表明,该序列拼接正确,SDS－PAGE显示,用重组的pQE30转化的M15菌经诱导后,有相对分子质量（M）约为52000的外源蛋白表达,Ni-NTA树脂纯化的表达产物纯度大于90％,因此,成功地构建了人源抗TNFa的ScFv基因,并在E.coli DH5a中表达和纯化的该基因的产物。     

Identification of the plasmid-encoded immunity protein for colicin El in the inner membrane of Escherichia coli

A set of plasmids containing portions of the Col El plasmid were transformed into recA⁻ cells. These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins. UV-irradiated cells label a 14.5 kDa band if they are phenotypically immune to colicin E1, and do not contain this band if they are sensitive to colicin E1. We conclude that the 14.5 kDa protein is the colicin E1 immunity protein. When the inner and outer membranes of these cells are fractionated, the labelled band appears in the inner membrane. The immunity protein must be an intrinsic inner membrane protein, confirming the predictions made by hydrophobicity calculations from primary sequence data.MaxicellCol El plasmidImmunity proteinHydrophobicity calculation     

Isolation and genetic characterization of the porcine apolipoprotein E gene

The present report describes the isolation and genetic characterization of the porcine apolipoprotein E (ape-E) gene. A single positive recombinant phage clone containing a 10·7-kb insert was isolated from a porcine genomic library, and a 4·2-kb fragment was subcloned and sequenced. The 4·2-kb fragment contained the entire apo-E gene in addition to upstream and downstream sequences (GenBank accession no. 470240). The porcine apo-E gene is made up of four exons and three introns, and encodes a preapo-E protein comprised of a signal peptide of 18 amino acids and a mature protein of 299 amino acids. The porcine apo-E gene contains a (CG)₁₃ microsatellite marker within intron three. This microsatellite is moderately polymorphic, and at least four alleles were evident at this locus among 10 animals from each of the Yorkshire, Hampshire, Landrace and Duroc breeds. Finally, localization of the porcine apo-E gene to chromosome 6 band q2·1 was determined by fluorescent in situ hybridization and confirmed by genetic linkage analysis.     

Cloning and expression in Escherichia coli of a recA-like gene from the acidophilic autotroph Thiobacillus ferrooxidans

A recombinant plasmid, pRSR100, containing the functional analogue of the Escherichia coli recA gene was isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. The plasmid complemented defects in DNA repair and homologous recombination in E. coli recA mutant strains. Antiserum raised against E. coli RecA protein reacted with the native but defective E. coli HB101 RecA protein; it did not react with protein extracts from the recA deletion mutant E. coli JK696, but it reacted with two protein bands in extracts of E. coli JK696(pRSR100). A single band with an apparent Mr equal to the higher-Mr band in E. coli JK696(pRSR100) was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum.     

Identification of the gene encoding transcription factor NusG of Thermus thermophilus.

The nusG gene of Thermus thermophilus HB8 was cloned and sequenced. It is located 388 bp downstream from tufB, which is followed by the genes for ribosomal proteins L11 and L1. No equivalent to secE preceding nusG, as in Escherichia coli, could be detected. The nusG gene product was overproduced in E. coli. A rabbit antiserum raised against the purified recombinant NusG reacted exclusively with one protein band of T. thermophilus crude extracts in Western blot (immunoblot) analyses, and no cross-reaction of the antiserum with E. coli NusG was observed. Recombinant NusG and the reacting T. thermophilus wild-type protein had identical sizes on sodium dodecyl sulfate-polyacrylamide gels. T. thermophilus and E. coli NusG have 45% identical and 22.5% similar amino acids, and similarities between the two proteins are most pronounced in carboxy-terminal regions. The T. thermophilus nusG gene could not rescue a nusG-deficient E. coli mutant strain.     

大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PA     

富含蛋氨酸玉米醇溶蛋白在大肠杆菌中的表达

目的:克隆玉米中富含蛋氨酸的10kD玉米醇溶蛋白,证明植物源目的基因在大肠杆菌中能够表达.方法:从玉米胚乳中克隆高蛋氨酸基因zein,通过PCR扩增zein片段,连接pGEX - 4T -1原核表达载体,转入大肠杆菌中,IPTG诱导后,HPLC测定蛋氨酸含量.结果:经PCR扩增出467bp条带,Blast分析同源性99%,经IPTG诱导进行SDS - PAGE检测,发现在36kD处出现一条明显的条带.诱导后菌体总蛋氨酸含量比正常菌体提高了9.6%.结论:证实了植物源10kD玉米醇溶蛋白在大肠杆菌中能够表达.     

Isolation of the Rhizobium leguminosarum NodF nodulation protein: NodF carries a 4''-phosphopantetheine prosthetic group.

Rhizobium species produce a protein product of the nodF gene that has a limited but recognizable homology to the well-characterized acyl carrier protein (ACP) of Escherichia coli. NodF functions together with NodE in generating a host-specific response to the plant host in the interchange of signals leading to the effective nodulation of roots (H.P. Spaink, J. Weinman, M.A. Djordjevic, C.A. Wijffelman, R.J.H. Okker, and B. J.J. Lugtenberg, EMBO J. 8:2811-2818, 1989; B. Scheres, C. van de Wiel, A. Zalensky, B. Horvath, H. Spaink, H. van Eck, F. Zwartkruis, A.M. Wolters, T. Gloudemans, A. van Kammen, and T. Bisseling, Cell 60:281-294, 1990). The nodFE region of Rhizobium leguminosarum has been cloned into a multicopy plasmid and has been shown in R. leguminosarum to code for a flavonoid-inducible protein that is effectively labeled by radioactive beta-alanine added to the growth medium. After purification, the labeled protein migrates as a single band with an apparent molecular weight of 5,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, more rapidly than E. coli ACP. In contrast, in native gels the protein is resolved into two bands, both identified as NodF by analysis of the amino terminus and both migrating more slowly than E. coli ACP. Pulse-chase experiments with labeled beta-alanine suggested that the slower-moving band may be the precursor of the faster band. The NodF protein carries a 4'-phosphopantetheine as a prosthetic group. A NodF fusion protein under the control of the lac promoter is expressed in E. coli and is labeled with beta-alanine, indicating that it is recognized by the ACP synthase of E. coli. The ACP phosphodiesterase of E. coli, which catalyzes the release of phosphopantetheine from E. coli ACP, does not remove phosphopantetheine from NodF.     

乙内酰脲水解酶基因在大肠杆菌中的克隆表达

节杆菌BT801的乙内酰脲酶系能够水解5-苄基乙内酰脲生成L-苯丙氨酸,其中乙内酰脲水解酶负责乙内酰脲的水解开环。乙内酰脲水解酶的表达对于乙内酰脲酶的催化机制研究及氨基酸的生物不对称合成都具有重要意义。通过PCR技术扩增得到乙内酰脲水解酶基因(hyuH),置于表达载体pT221的,17启动子下游,将构建的重组质粒引入大肠杆菌BL21(DE3)。SDS-PAGE分析在相对分子量50kD处有一较强的表达带,经薄层扫描分析目的蛋白占全菌蛋白的40％,主要以可溶性形式存在,活性分析表明表达产物具有天然的酶活性。     

猪丹毒丝菌C43065株表面保护性抗原AN端保护区在大肠杆菌中的表达

目的：在大肠杆菌中表达猪丹毒丝菌C43065株表面保护性抗原A（SpaA）N端保护区（SpaA-N）,并检测其抗原性。方法：利用PCR方法从猪丹毒丝菌C43065株基因组中扩增出spaA基因片段,构建pMD18-spaA重组质粒并对插入片段进行测序;以pMD18-spaA重组质粒为模板,PCR扩增得到spaA-N基因片段,构建重组表达质粒pGEX-spaA-N,经序列测定证实正确后转化大肠杆菌BL21（DE3）,再经IPTG诱导表达GST-SpaA-N融合蛋白并纯化。结果：扩增得到的spaA基因长1881bp,编码由626个氨基酸残基构成的多肽;SDS-PAGE和Western印迹检测结果表明,诱导表达获得相对分子质量约64000的GST-SpaA-N融合蛋白,该融合蛋白能与相应抗体发生特异性反应。结论：获得了在大肠杆菌中可溶性表达的GST-SpaA-N融合蛋白,为进一步研究猪丹毒丝菌免疫保护性抗原奠定了基础。     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Regional assignment of two genes of the human branched-chain alpha-keto acid dehydrogenase complex: the E1 beta gene (BCKDHB) to chromosome 6p21-22 and the E2 gene (DBT) to chromosome 1p31

Maple syrup urine disease (MSUD) is caused by the deficiency of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The multienzyme complex is a macromolecule (Mr 4 X 10(6] consisting of at least six distinct subunits. In this study, the human E1 beta gene (BCKDHB) has been localized to human chromosome 6 by hybrid somatic cell analysis, and regionally assigned to chromosome bands 6p21-22 by in situ hybridization. The E2 gene (DBT), which was previously localized to chromosome 1, is regionally assigned to the chromosome band 1p31 also by in situ hybridization. Localization of the E1 beta gene to chromosome 6p21-22 assigns another major human disease locus to a region that contains several important genes, including the major histocompatability complex, tumor necrosis factor, and heat-shock protein HSP70. Mapping of the E1 beta and the E2 genes may provide information for the linkage analysis of MSUD families with mutations in these two loci.     

人源化抗肝癌单链抗体与牛蛙核糖核酸酶融合基因的构建及表达

利用RT-PCR的方法从牛蛙肝脏中克隆牛蛙核糖核酸酶(RC—RNase)基因并进行序列测定;将人源化抗肝癌单链抗体(scFv)基因与RC—RNase基因相连接,制备scFv—RC—RNase融合基因表达质粒,转化大肠杆菌B121(DE3),用IPTG诱导进行表达。以SDS-PAGE和免疫印迹法对表达产物进行分析鉴定。序列分析表明,扩增出的RC—RNase基因片断大小约为405bpc,经SDS—PAGE和免疫印迹分析显示,scFv—RC—RNase融合基因表达质粒在大肠杆菌中的诱导表达产物出现相对分子质量约为38000的一条新生蛋白带,与预期结果相符。融合蛋白表达量占菌体总蛋白量的18．5％,主要以包涵体形式存在。表明成功地构建了抗肝癌scFv—RC—RNase融合基因,并在大肠杆菌中获得有效表达,为进一步进行肝癌的导向治疗研究奠定了基础。     

猪丹毒丝菌C43311株spaA基因N端免疫保护区的克隆和表达

采用PCR从猪丹毒丝菌C43311株基因组中扩增出编码表面保护性抗原A的spaA基因片段,将其克隆到pMD18-T载体并对插入片段进行测序.用spaA基因N端免疫保护区(spaA-N)的特异引物从重组质粒pMD18-spaA中扩增得到spaA-N片段,将其定向插入原核表达载体pGEX-6p-2中,构建重组表达质粒pGEX-spaA-N,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白GST-SpaA-N,用SDS-PAGE和Western印迹检测表达产物.测序结果表明spaA基因片段大小为1881bp,与已报道的不同血清型菌株spaA基因的核苷酸序列比较,核苷酸序列同源性在93%～99%之间.SDS-PAGE结果显示表达蛋白约为64kDa,与预期的大小相近.Western印迹检测结果表明,表达的融合蛋白GST-SpaA-N能与C43311株SpaA蛋白的抗血清发生特异性反应,证明原核融合表达蛋白具有免疫反应性.     

乙型肝炎病毒C蛋白基因的克隆表达

根据已知的C蛋白基因序列设计一对引物,用PCR法从HBV adw亚型全基因组克隆中扩增出约500bp的NDA片段,将该基因克隆到表达质粒pQE30中,转化大肠杆菌,进一步对重组质粒中的插入片段进行DNA测序,证明是C基因,阳性克隆子经IPTG诱导后,获得了目的蛋白的表达,菌体经超声破碎后,目的蛋白主要存在于上清中,为下一步研究其抗原性和免疫原性奠定了基础。