Rapid and efficient isolation of highly specific antibodies from an antiserum against a pool of proteins

Antibodies with desired specificity to proteins of interest provide important and versatile tools for detecting and localizing specific proteins in organisms. With the rapidly increasing number of genes cloned, the demand for antibodies to the gene products is increasing greatly. We developed a procedure to isolate highly specific antibodies to an insect intestinal mucin (IIM) from a polyclonal antiserum, which served as a “library of antibodies,” by using an E. coli lysate of the IIM cDNA clone. This procedure allows rapid and efficient isolation of target protein specific antibodies from a polyclonal antiserum made against a pool of antigens.     

Immunological quantification of recA protein in cell extracts ofE. coli after exposure to chemical mutagens or UV radiation

Increased synthesis of RecA protein is induced inE. coli cells after their damage, the rate of synthesis being dependent on the extent of DNA alterations. The level of the RecA protein was determined inE. coli cell extracts after damage induced by NQO, MNNG, MMC, NAL or UV radiation, using competitive enzyme-linked immunosorbent assay (ELISA). PurifiedE. coli RecA protein and rabbit monospecific polyclonal antibodies against it were prepared for the quantitative assay. The level of theRecA protein was increased after treatment with all mutagens. Contrary to other induced proteins, the synthesis of the RecA protein increased within 30 min after damage with UV radiation at a relatively slow rate. The ELISA method made it possible to determine 0.5–50 ng of the RecA protein in bacterial extracts. The method can be employed as an auxiliary test for DNA damage determination and also in studied concerning the role of the RecA protein in repair processes. Translated by I. Miler     

The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid

Shufflon DNA rearrangement selects one of seven PilV proteins with different C-terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64. The PilV proteins determine the recipient specificity in liquid matings. A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA′, -C, and -C′ proteins, while E. coli B was recognized only by the PilVA′ protein. To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E. coli K-12 waa (rfa) mutants and E. coli B transformants as recipient cells. E. coli K-12 waa mutants lack receptors for specific PilV proteins. E. coli B cells carrying waaJ or waaJKL genes of E. coli K-12 were recognized by donors expressing the PilVC′ protein or the PilVC and -C′ proteins, respectively, in addition to the PilVA′ protein. Addition of E. coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings. We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. Received: 2 September 1999 / Accepted: 18 November 1999     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Promotion of Bacteriophage Induction and Recombination by the Cloned Bordetella pertussis recA Gene Is Copy-Number Dependent

Favre and coworkers (Favre et al., Biochimie 73:235–244, 1991) previously reported that the Bordetella pertussis recA gene present at high copy number could promote a low frequency of recombination, but not bacteriophage induction in Escherichia coli RecA⁻ mutants. Reexamination of these phenotypes demonstrated that, in contrast to the previous study, when this gene is present at high copy number, it can stimulate a 2- to 4-log frequency of bacteriophage induction in the presence of mitomycin C, but no appreciable spontaneous induction. The cloned gene, whether it was present in high or low copy number, also promoted a low frequency of intrachromosomal recombination of two duplicated genes in Escherichia coli. These results suggest that a high concentration of the B. pertussis RecA protein is required to promote high-frequency mitomycin C-stimulated bacteriophage induction, but it facilitates intrachromosomal recombination at a very low frequency in E. coli RecA⁻ mutants. The ability of the B. pertussis RecA protein to promote mitomycin C induction and its inability to appreciably stimulate spontaneous induction of bacteriophage suggest that this protein possesses a unique phenotype compared with other RecA proteins.     

Production of Polyclonal Antibodies to the Recombinant Potato virus M (PVM) Non‐structural Triple Gene Block Protein 1 and Coat Protein

The genes encoding the coat protein (CP) and triple gene block protein 1 (TGBp1) of Potato virus M (PVM) were cloned into expression vector pET‐45b(+) (N‐terminal 6xHis tag) and expressed in E. coli Rosetta gami‐2(DE3). The purified recombinant antigens were used for raising polyclonal antibodies. The antibodies against recombinant CP were successfully used in Western blot analysis, plate‐trapped ELISA and DAS‐ELISA as a coating for PVM detection in infected potato leaf samples. The antibodies against recombinant non‐structural protein detected the TGBp1 only in Western blot analysis. This is the first report of the production of polyclonal antibodies against recombinant coat protein and TGBp1 of PVM and their use for detecting the virus.     

Four Rice Seed cDNA Clones Belonging to the α-Amylase/Trypsin Inhibitor Gene Family Encode Potential Rice Allergens

Four rice seed proteins encoded by cDNAs belonging to the α-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial celllysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.     

Cloning and characterization of recA genes froM Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r

The recA genes of Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r have been isolated and introduced into Escherichia coli K-12. All the heterologous genes restore resistance to killing by UV irradiation and the mutagen 4-nitroquinoline-1-oxide in RecA- E. coli K-12 hosts. Recombination proficiency is also restored as measured by formation of Lac+ recombinants from duplicated mutant lacZ genes and the ability to propagate phage lambda derivatives requiring host recombination functions for growth (Fec-). The cloned heterologous genes increase the spontaneous induction of lambda prophage in lysogens of a recA strain. Addition of mitomycin C stimulates phage production in cells carrying the E. coli B/r and S. flexneri recA genes, but little or no stimulation is seen in cells carrying the E. carotovora and P. vulgaris recA genes. After treatment with nalidixic acid, the heterologous RecA proteins are synthesized at elevated levels, a result consistent with their regulation by the E. coli K-12 LexA repressor. Southern hybridization and preliminary restriction analysis indicate divergence among the coding sequences, but antibodies prepared against the E. coli K-12 RecA protein cross-react with the heterologous enzymes, indicating structural conservation among these proteins.     

Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS—polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.     

Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.     

Effect of a lexA41(Ts) mutation on DNA repair in recA(Def) derivatives of Escherichia coli K-12

Summary Derivatives of Escherichia coli K-12 carrying a deletion of the recA gene survive exposure to UV (254 nm) better if they also contain the lexA41 mutation which codes for a labile LexA protein. This effect of the lexA41 mutation is not observed in comparable strains carrying a uvr A6 mutation. Using two independent methods to detect pyrimidine dimers we found that UV irradiated RecA deficient cells removed dimers from their DNA more rapidly if they contained the lexA41 mutation than if the contained the wild-type lexA gene. Our results are consistent with the idea that a relatively high level of UvrABC incision nuclease resulting from inefficient repression of the corresponding genes by the labile LexA41 protein facilitates excision of pyrimidine dimers from the DNA of UV irradiated cells.     

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [³⁵ S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.     

Production of polyclonal antibodies to a recombinant coat protein of potato virus Y

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Ω. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.     

Ectopic Gene Conversions in Four <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> Genomes: Increased Recombination in Pathogenic Strains

We characterized the ectopic gene conversions in the genomes of the K-12 MG1655, O157:H7 Sakai, O157:H7 EDL933, and CFT073 strains of E coli. Compared to the three pathogenic strains, the K-12 strain has a much smaller number of gene families, its gene families contain fewer genes, and gene conversions are less frequent. Whereas the three pathogenic strains have gene conversions covering hundreds of nucleotides when their flanking regions have as little as 50% similarity, flanking region similarity of at least 94% on both sides of the converted region is required to observe conversions of more than 87 nucleotides in the K-12 strain. Recombination is therefore more frequent and requires less sequence similarity in the three pathogenic strains than in K-12. This higher recombination level might be due to mutations in some of their mismatch-repair genes. In contrast with the gene conversions present in the yeast genome, the gene conversions found in the E. coli genomes do not occur more frequently between duplicated genes that are close to one another than between duplicated genes that are far apart and are randomly distributed along the length of the genes. In E. coli, gene conversions are not more frequent near the origin of replication. However, they do occur more frequently near the terminus of replication of the Sakai genome, where multigene family members are more abundant. This suggests that, in E. coli, gene conversions occur randomly between genes located in different chromosomal locations or located on different copies of the multiple chromosomes found in E. coli cells.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Initial function analysis of a novel erythroid differentiation related geneEDRF1

Erythroid differentiation depends on the establishment of specific patterns of gene expression. Hypersensitive site 2 (HS2, serving as a major enhancer of globin genes)-binding proteins may be involved in its natural open chromosomal environment formation. Previously we prepared monoclonal antibodies against HS2-binding nuclear proteins of terminal differentiated erythroid cells. By utilizing the monoclonal antibodies, we screened λ-gt11 human fetal liver cDNA expression library and obtained one cDNA clone, which was named erythroid differentiation related gene (EDRF1, Genbank accession number AF040247), encompassing an entire open reading frame. We investigated the expression pattern ofEDRF1 by RT-PCR technique. And a clue to the function ofEDRF1 has been found from confirmation of high levels ofEDRF1 mRNA in differentiated K562 and human fetal liver tissue. To illuminate the function ofEDRF1 in K562 cells, sense and antisenseEDRF1 constructs were prepared and transfected into K562 cells. α-globin mRNA was down-regulated and EpoR (erythropoietin receptor) mRNA expression was increased in antisense transfected cells. Cells transfected with sense construct grew more slowly than control cells suggested by [³H] thimidine incorporation experiments. Suppression of K562 proliferation was accompanied by increased spontaneous hemoglobin synthesis demonstrated by spectrometry. K562 cells transfected with sense construct exhibited reduced clongenicity compared with control cells in methycellulose culture. These data provided the evidence thatEDRF1 can influence globin expression and hemoglobin synthesis in K562 cells and modulated self-renewal in K562 cells.     

Initial function analysis of a novel erythroid differentiation related gene EDRF1

Erythroid differentiation depends on the establishment of specific patterns of gene expression. Hypersensitive site 2 (HS2, serving as a major enhancer of globin genes)-binding proteins may be involved in its natural open chromosomal environment formation. Previously we prepared monoclonal antibodies against HS2-binding nuclear proteins of terminal differentiated erythroid cells. By utilizing the monoclonal antibodies, we screened λ-gt11 human fetal liver cDNA expression library and obtained one cDNA clone, which was named erythroid differentiation related gene (EDRF1, Genbank accession number AF040247), encompassing an entire open reading frame. We investigated the expression pattern ofEDRF1 by RT-PCR technique. And a clue to the function ofEDRF1 has been found from confirmation of high levels ofEDRF1 mRNA in differentiated K562 and human fetal liver tissue. To illuminate the function ofEDRF1 in K562 cells, sense and antisenseEDRF1 constructs were prepared and transfected into K562 cells. α-globin mRNA was down-regulated and EpoR (erythropoietin receptor) mRNA expression was increased in antisense transfected cells. Cells transfected with sense construct grew more slowly than control cells suggested by [³H] thimidine incorporation experiments. Suppression of K562 proliferation was accompanied by increased spontaneous hemoglobin synthesis demonstrated by spectrometry. K562 cells transfected with sense construct exhibited reduced clongenicity compared with control cells in methycellulose culture. These data provided the evidence thatEDRF1 can influence globin expression and hemoglobin synthesis in K562 cells and modulated self-renewal in K562 cells.     

黄瓜过敏性诱导反应蛋白基因(CsHIR1)原核表达及其多克隆抗体制备

该试验用黄瓜霜霉菌侵染黄瓜幼苗,并通过PCR方法克隆其过敏性诱导反应蛋白(HIR)的基因CsHIR1,构建原核表达载体pET28a-CsHIR1,实现在大肠杆菌(E.coli)BL21(DE3)中的高效表达;对诱导表达的时间和IPTG的浓度进行了优化;利用钴离子螯合层析纯化了重组蛋白并制备高效价多克隆抗血清。结果表明:该黄瓜过敏性反应诱导蛋白以包涵体的形式表达,最佳诱导时间和IPTG浓度分别为4h和0.5mmol·L-1;经纯化,得到高纯度的分子量为34kD重组蛋白CsHIR1。Western blotting显示CsHIR1的抗体具有较好的特异性。原核表达体系的建立和多克隆抗体的制备为进一步研究CsHIR1基因在黄瓜中的功能奠定了基础。