DNA重组酶FLP原核表达与多步法纯化及其活性检测

DNA重组酶FLP存在于酵母2μ质粒上,能识别34bp的FRT位点,并根据2个FRT位点的相对方向完成位点间DNA序列的交换、重组、删除与逆转,在现代分子生物学理论研究与基因工程技术开发中具有广泛应用。构建了在原核大肠杆菌中高效表达FLP重组酶的表达载体pQE32-flpe并建立起相应的原核高效表达体系,在原核细菌大肠杆菌M15菌株中实现FLP酶蛋白的高效表达,同时建立了相应的纯化方法。纯化时先用硫酸铵沉淀法富集FLP酶蛋白,经透析脱盐后再用镍离子鳌合微柱(0.5~1.0mL)亲合层析梯度洗脱的方法获得纯化的FLP酶蛋白。通过构建含有2个方向相同的FRT序列位点的质粒pUC18-FRT-gfp-FRT和含有1个FRT位点的表达载体pET30a-FRT,并分别以其为底物来检测FLP重组酶的删除、交换与重组功能的活性。结果表明,该方法不仅能有效表达FLP酶蛋白,并能行之有效地纯化FLP酶蛋白,以及检测纯化的FLP酶蛋白对DNA序列的删除、重组与交换功能。该方法简单易行并能获得有活性的FLP酶蛋白,为深入研究其机理以及研发相应的DNA重组技术提供重要参考。     

Purification and characterization of the single-stranded DNA binding protein from Streptococcus pneumoniae

The Escherichia coli single-stranded DNA binding (SSB) protein is a non-sequence-specific DNA binding protein that functions as an accessory factor for the RecA protein-promoted three-strand exchange reaction. An open reading frame encoding a protein similar in size and sequence to the E. coli SSB protein has been identified in the Streptococcus pneumoniae genome. The open reading frame has been cloned, an overexpression system has been developed, and the protein has been purified to greater than 99% homogeneity. The purified protein binds to ssDNA in a manner similar to that of the E. coli SSB protein. The protein also stimulates the S. pneumoniae RecA protein and E. coli RecA protein-promoted strand exchange reactions to an extent similar to that observed with the E. coli SSB protein. These results indicate that the protein is the S. pneumoniae analog of the E. coli SSB protein. The availability of highly-purified S. pneumoniae SSB protein will facilitate the study of the molecular mechanisms of RecA protein-mediated transformational recombination in S. pneumoniae.     

The gamma protein specified by bacteriophage gamma. Structure and inhibitory activity for the recBC enzyme of Escherichia coli.

The protein encoded by the gam gene of bacteriophage lambda ("gamma protein") is a specific inhibitor of the recBC enzyme of Escherichia coli. The lambda protein has been purified approximately 2,000-fold, and its structure and inhibitory activity have been characterized. It appears to be composed of two identical subunits of 16,500 daltons, inhibits all of the catalytic activities of the recBC enzyme with apparently equal efficiency, but has no effect upon any other E. coli or lambda-DNase tested. Inhibition does not occur unless recBC enzyme is exposed to gamma protein prior to reaction of the enzyme with DNA. The inhibitory activity is independent of temperature, and no catalytic activity has been detected that might fulfill the inhibitory function. It appears instead that the inhibition involves a stoichiometric, rather than a catalytic interaction between gamma protein and the enzyme. Reaction kinetics for the recBC enzyme inhibited by gamma protein show no anomalous protein--only a depressed rate. Inhibition is not competitive and does not appear to affect the enzyme's affinity for DNA. The enzyme remains inhibited after it is separated from "excess" gamma protein by gel filtration or sedimentation in a glycerol gradient, and inhibited enzyme has a reduced electrophoretic mobility compared to that of uninhibited enzyme. Gamma Protein inhibits recBC enzyme which has been reconstituted from cell-free extracts by complementation in vitro, but at least one of the complementing factors present in extracts from recB- cells does not by itself form a complex with gamma protein. The mechanism of inhibition and the implications of these results from gamma replication and recombination are discussed.     

Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences.

The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro. We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo. One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein. This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence. The second mutation lies within the 8-bp core region of the FLP target sequence. The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange. The mutation in the core region abolishes recombination with a wild-type site. However, recombination between two mutated sites is very efficient. This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.     

Biochemical and immunological characterization of the DNA binding protein (RBP-J kappa) to mouse J kappa recombination signal sequence.

We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E. coli and unable to bind J kappa-RS. Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].     

Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein

A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.     

Purification and characterization of the gene 1.2 protein of bacteriophage T7

Gene 1.2 of bacteriophage T7, located near the primary origin of DNA replication at position 15.37 on the T7 chromosome, encodes a 10,059-dalton protein that is essential for growth on Escherichia coli optA1 strains (Saito, H., and Richardson, C. C. (1981) J. Virol. 37, 343-351). In the absence of the T7 1.2 and E. coli optA gene products, the degradation of E. coli DNA proceeds normally, and T7 DNA synthesis is initiated at the primary origin. However, T7 DNA synthesis ceases prematurely and the newly synthesized DNA is degraded; no viable phage particles are released. The gene 1.2 protein has been purified to apparent homogeneity from cells in which the cloned 1.2 gene is overexpressed. Purification of the [35S] methionine-labeled protein was followed by monitoring the radioactivity of the protein and by gel electrophoresis. The purified protein has been identified as the product of gene 1.2 on the basis of molecular weight and partial amino acid sequence. We have found that extracts of E. coli optA1 cells infected with T7 gene 1.2 mutants are defective in packaging exogenous T7 DNA when such extracts are prepared late in infection. Purified gene 1.2 protein restores packaging activity to these defective extracts, thus providing a biological assay for gene 1.2 protein. No specific enzymatic activity has been found associated with the purified gene 1.2 protein.     

The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene

Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity.     

DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site

The 2 mu plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombination system consisting of the FLP protein and two inverted recombination sites on the plasmid. The minimal fully functional substrate for in-vitro recombination in this system consists of two FLP protein binding sites separated by an eight base-pair spacer sequence. We have used site-directed mutagenesis to generate every possible mutation (36 in all) within 11 base-pairs of one FLP protein binding site and the base-pair immediately flanking it. The base-pairs within the binding site can be separated into three classes on the basis of these results. Thirty of the 36 sequence changes, including all three at seven different positions (class I) produce a negligible or modest effect on FLP protein-promoted recombination. In particular, most transition mutations are well-tolerated in this system. In only one case do all three possible mutations produce large effects (class II). At three positions, clustered near the site at which DNA is cleaved by FLP protein, one of the two possible transversions produces a large effect on recombination, while the other two changes produce modest effects (class III). For seven mutants for which FLP protein binding was measured, a direct correlation between decreases in recombination activity and in binding was observed. Positive effects on the reaction potential of mutant sites are observed when the other FLP binding site in a single recombination site is unaltered or when the second recombination site in a reaction is wild-type. This suggests a functional interaction between FLP binding sites both in cis and in trans. When two mutant recombination sites (each with 1 altered FLP binding site) are recombined, the relative orientation of the mutations (parallel or antiparallel) has no effect on the result. These results provide an extensive substrate catalog to complement future studies in this system.     

The minimal duplex DNA sequence required for site-specific recombination promoted by the FLP protein of yeast in vitro.

The 2-micron plasmid of the yeast Saccharomyces cerevisiae codes for a site-specific recombinase ('FLP') that efficiently catalyses recombination across the plasmid's two 599 bp repeats both in vivo and in vitro. We have used the partially purified FLP protein to define the minimal duplex DNA sequence required for intra- and intermolecular recombination in vitro. Previous DNase footprinting experiments had shown that FLP protected 50 bp of DNA around the recombination site. We made BAL31 deletions and synthetic FLP sites to show that the minimal length of the site that was able to recombine with a wild-type site was 22 bp. The site consists of two 7 bp inverted repeats surrounding an 8 bp core region. We also showed that the deleted sites recombined with themselves and that one of three 13 bp repeated elements within the FLP target sequence was not necessary for efficient recombination in vitro. Mutants lacking this redundant 13 bp element required a lower amount of FLP recombinase to achieve maximal yield of recombination than the wild type site. Finally, we discuss the structure of the FLP site in relation to the proposed function of FLP recombination in copy number amplification of the 2-micron plasmid in vivo.     

Purification and characterization of the RecA protein from Streptococcus pneumoniae

Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 60% identical to that of the RecA protein from Escherichia coli. We have developed an overexpression system for the S. pneumoniae RecA protein and have purified the protein to greater than 99% homogeneity. The S. pneumoniae RecA protein has ssDNA-dependent NTP hydrolysis and NTP-dependent DNA strand exchange activities that are generally similar to those of the E. coli RecA protein. In addition to its role as a DNA recombinase, the E. coli RecA protein also acts as a coprotease, which facilitates the cleavage and inactivation of the E. coli LexA repressor during the SOS response to DNA damage. Interestingly, the S. pneumoniae RecA protein is also able to promote the cleavage of the E. coli LexA protein, even though a protein analogous to the LexA protein does not appear to be present in S. pneumoniae.     

The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases

DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.     

Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair.

The incision of damaged DNA by the Escherichia coli UvrABC endonuclease requires ATP hydrolysis. Although the deduced sequence of the UvrB protein suggests a putative ATP binding site, no nucleoside triphosphatase activity is demonstrable with the purified UvrB protein. The UvrB protein is specifically proteolyzed in E. coli cell extracts to yield a 70 kD fragment, referred to as UvrB*, which has been purified and is shown to possess a single-strand DNA dependent ATPase activity. Substrate specificity and kinetic analyses of UvrB* catalyzed nucleotide hydrolysis indicate that the stimulation in DNA dependent ATPase activity following formation of the UvrAB complex results from the activation of the normally sequestered UvrB associated ATPase. Using nucleotide analogues, it can be shown that this activity is essential to the DNA incision reaction carried out by the UvrABC complex.     

Postinfection control by bacteriophage T4 of Escherichia coli recBC nuclease activity.

Infection by bacteriophage T4 has previously been shown to cause a rapid inhibition of the host recBC DNase, an ATP-dependent DNase that is required for genetic recombination in Escherichia coli. We report here the partial purification of a protein ("T4 rec inhibitor") from extracts of T4-infected cells and some characteristics of the in vitro inhibition reaction with purified inhibitor and recBC nuclease. This inhibitory activity could not be purified from extracts of uninfected E. coli. Both the ATP-dependent exonuclease and DNA-dependent ATPase activities of recBC DNase are inhibited by T4 rec inhibitor. Experiments suggest that the inhibitor interacts with the nuclease in a stoichiometric manner. The biological significance of this inhibition is discussed with respect to control reactions in phage-infected cells.     

The histone-like H protein of Escherichia coli is ribosomal protein S3.

We report the purification of four proteins from Escherichia coli that stimulate or inhibit inter- and/or intramolecular recombination promoted by the yeast plasmid-encoded FLP protein. The proteins are identified as the ribosomal proteins S3 (27 kDa), L2 (26 kDa), S4 (24 kDa), and S5 (16 kDa), on the basis of N-terminal sequence analysis. The S3 protein is found to be identical to H protein, an E. coli histone-like protein that is related to histone H2A immunologically and by virtue of amino acid content. The H protein/S3 identity is based on co-migration on polyacrylamide gels, heat stability, amino acid analysis, and effects on FLP-promoted recombination. These results are relevant to current studies on the structure of the E. coli nucleoid. Since the H protein has previously been found associated with the E. coli nucleoid, the results indicate that either (a) some ribosomal proteins serve a dual function in E. coli, or, more likely, (b) ribosomal proteins can and are being mis-identified as nucleoid constituents.     

The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.     

Nucleotide sequence and expression of the gene for the site-specific integration protein from bacteriophage HP1 of Haemophilus influenzae.

The nucleotide sequence of the leftmost 2,363 base pairs of the HP1 genome, which includes the attachment site (attP) and the integration region, was determined. This sequence contained an open reading frame encoding a 337-residue polypeptide, which is a member of the integrase family of site-specific recombination proteins as judged by sequence comparison. The open reading frame was located immediately adjacent to the att site and was oriented so that initiation of translation would begin distal to the att site and end in its immediate vicinity. Expression of this DNA segment in Escherichia coli provided extracts which promoted site-specific recombination between plasmids containing cloned HP1 attP and Haemophilus influenzae attB sites. This recombination was directional, since no reaction was observed between plasmids containing attR and attL sites. The reaction was stimulated by the accessory protein integration host factor of E. coli. Evidence was also obtained that the integration host factor influenced the levels of HP1 integrase expression. The deduced amino acid sequence of HP1 integrase has remarkable similarity to that deduced for the integrase of coliphage 186.     

A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V

Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nfi gene. A. fulgidus endonuclease V was cloned and expressed in E. coli as a C-terminal hexa-histidine fusion protein. The C-terminal fusion protein was purified to apparent homogeneity by a combination of Ni(++) affinity and MonoS cation exchange liquid chromatography. The purified C-terminal fusion protein has a molecular weight of about 25kDa and showed endonuclease activity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg(2+) and an optimum reaction temperature at 85 degrees C. However, in contrast to E. coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosine. These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution.