Expression and purification of codon-optimized cre recombinase in E. coli.

The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the widely used systems for selectable marker gene removal. We have overexpressed codon-optimized cre gene in pColdIV and pET28a(+) vector systems and purified His6-Cre recombinase by immobilized metal affinity chromatography. N-terminal His6 tagged Cre recombinase obtained was approximately 26 fold purified and promoted the site-specific recombination of two loxP sites of linearized pLox2+ vector allowing the excision of a re-circularized plasmid and a short stretch of DNA containing the recombined loxP site. The results of the expression using two vectors, purification and activity assessment of His6 tagged Cre recombinase is presented here.

Purification and In Vitro Characterization of Zinc Finger Recombinases.

Zinc finger recombinases (ZFRs) are designer site-specific recombinases that have been adapted for a variety of genome editing purposes. Due to their modular nature, ZFRs can be customized for targeted sequence recognition and recombination. There has been substantial research on the in vivo properties and applications of ZFRs; however, in order to fully understand and customize them, it will be necessary to study their properties in vitro. Experiments in vitro can allow us to optimize catalytic activities, improve target specificity, measure and minimize off-target activity, and characterize key steps in the recombination pathway that might be modified to improve performance. Here, we present a straightforward set of protocols for the expression and purification of ZFRs, an assay system for catalytic proficiency in vitro and bandshift assays for detection of sequence-specific DNA interactions.

Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family.

Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF). Here we present a set of protocols for the overexpression and purification of bacteriophage ϕC31 and Bxb1 integrase and RDF proteins, their use in DNA assembly reactions, and subsequent modification of the resulting DNA assemblies.

Functional expression and purification of Anabaena PCC 7120 XisA protein.

Anabaena PCC 7120 xisA gene product mediates the site-specific excision of 11,278 bp nifD element in heterocysts formed under nitrogen starvation conditions. Although XisA protein possesses both site-specific recombinase and endonuclease activities, till date neither xisA transcript nor XisA protein has been detected. Gene encoding XisA protein was isolated from plasmid pMX25 and overexpressed in Escherichia coli BL21 DE3 yielding 7.7 mg enzyme per L of growth culture in soluble fraction. His-tagged XisA was purified using Ni-NTA affinity chromatography with 95% recovery. The purified XisA showed a single band on SDS-PAGE with molecular mass of 52 kDa. Identity of XisA was confirmed by MALDI-TOF analysis and functionality of enzyme was confirmed using restriction digestion. A PCR based method was developed to monitor excision by XisA, which displayed near 100% activity in E. coli within 1 h at 37 (°)C on LB under static condition.

The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system.

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to be a site-specific tyrosine recombinase that acts with the 36-bp recombination site mrpS. The present report gives a comprehensive characterization of the composition as well as the position of the spacer and MrpA binding sites within mrpS. Experiments revealed a spacer consisting of 6 remarkably variable nucleotides in the middle of the mrpS-site. A reduction in the spacer to 5 nucleotides abolished recombination. Investigation of the MrpA binding sites showed the importance of its 15 nucleotides on an effective recombination. Among almost randomly exchangeable nucleotides, two nucleotides were identified as essential for MrpA binding. Alteration of either of these nucleotides led to a reduction in MrpA binding down to 2 % or even to no binding. Based on these results, a new left element/right element (LE/RE) deletion system was developed. The constructed heteromeric mrpS-sites are efficiently resolved by MrpA. The resulting double mutated (LE/RE) site can no longer be used as a recombination site by MrpA. The system has been successfully applied for the generation of multiple-targeted deletions in the genome of E. coli.

Genetic engineering of mammalian cells by direct delivery of FLP recombinase protein.

Protein transduction is based on the ability of certain peptides, designated as cell penetrating peptides (CPPs), to intracellularly deliver cargo molecules, such as peptides and proteins. In combination with site specific recombination, CPP-mediated delivery of recombinases enables a precise and highly efficient control of gene expression in cultured cells and mice. Herein, we provide detailed protocols for engineering and purification of a cell-permeant FLP recombinase protein. Two examples describe the use of cell permeant FLP for excising prespecified fragments from transgenes expressed in fibroblasts and mouse embryonic stem cells. A third example describes the combined use of cell-permeant Cre and FLP recombinases to reversibly induce transgenes in embryonic stem cells. We anticipate that the protocols described herein will be widely used for various genetic interventions addressing complex biological questions.

Purification and characterization of bacteriophage P22 Xis protein.

The temperate bacteriophages lambda and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. lambda and P22 Xis proteins are both small proteins (lambda Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for lambda Xis, 11.16). However, the P22 Xis and lambda Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the attP arms containing the major Xis binding sites from bacteriophages lambda, P22, L5, HP1, and P2 and the conjugative transposon Tn916. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.

The tra region of the conjugative plasmid pIP501 is organized in an operon with the first gene encoding the relaxase.

The tra genes orf1 to orf11 of pIP501 were shown to be transcribed as a single operon of 11.3 kb in Enterococcus faecalis by reverse transcription-PCR. The transcriptional start site of the tra mRNA was mapped at 110 bp upstream from the predicted TTG start codon of the first gene of the operon, the traA relaxase. The TraA protein (660 amino acids) and a C-terminally truncated version of the TraA protein (293 amino acids) were purified as fusions with glutathione S-transferase. oriT cleavage activity of both TraA proteins was demonstrated in vitro on supercoiled plasmid pVA2241 DNA containing oriT(pIP501). The activity of the DNA relaxase TraA is strictly dependent on the presence of Mg(2+) or Mn(2+) and is highest at temperatures of between 42 and 45C.

Purification and characterization of the R64 shufflon-specific recombinase.

The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Binding of the purified integron DNA integrase Intl1 to integron- and cassette-associated recombination sites.

The site-specific recombinase Intl1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron-associated attl1 site and the 59-base element (59-be) family of sites that are associated with gene cassettes. Intl1 includes the four conserved amino acids that are characteristic of members of the integrase family, and Intl1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. Intl1 was purified as a fusion protein and shown to bind to isolated attl1 or 59-be recombination sites. Binding to attl1 was considerably stronger than to a 59-be. Binding adjacent to the recombination cross-over point was not detected. A strong Intl1 binding site within attl1 was localized by both deletion and footprinting analysis to a 14 bp region 24-37 bp to the left of the recombination cross-over point, and this region is known to be critical for recombination in vivo (Recchia et al., 1994). An imperfect (13/15) direct repeat of this region, located 41-55 bp to the left of the recombination cross-over point, contains a weaker Intl1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding.

Distribution of the Rad51 recombinase in human and mouse spermatocytes.

In vitro, the human Rad51 protein (hRad51) promotes homologous pairing and strand exchange reactions suggestive of a key role in genetic recombination. To analyse its role in this process, polyclonal antibodies raised against hRad51 were used to study the distribution of Rad51 in human and mouse spermatocytes during meiosis I. In human spermatocytes, hRad51 was found to form discrete nuclear foci from early zygotene to late pachytene. The foci always co-localized with lateral element proteins, components of the synaptonemal complex (SC). During zygotene, the largest foci were present in regions undergoing synapsis, suggesting that Rad51 is a component of early recombination nodules. Pachytene nuclei showed a greatly reduced level of Rad51 labelling, with the exceptions of any asynapsed autosomes and XY segments, which were intensely labelled. The distribution of Rad51 in mouse spermatocytes was similar to that found in human spermatocytes, except that in this case Rad51 was detectable at leptotene. From these results, we conclude that the Rad51 protein has a role in the interhomologue interactions that occur during meiotic recombination. These interactions are spatially and temporally associated with synapsis during meiotic prophase I.

Tn5 transposase mutants that alter DNA binding specificity.

Tn5 transposase (Tnp) binds to Tn5 and IS50 end inverted repeats, the outside end (OE) and the inside end (IE), to initiate transposition. We report the isolation of four Tnp mutants (YH41, TP47, EK54 and EV54) that increase the OE-mediated transposition frequency and enhance the binding affinity of Tnp for OE DNA. In addition, two of the Tnp mutants (TP47 and EK54) appear to be change-of-specificity mutants, since they alter the recognition of OE versus IE relative to the wild-type Tnp. EK54 enhances OE recognition but decreases IE recognition. TP47 enhances both OE and IE recognition but with a much greater enhancement for IE than for OE. This change-of-specificity effect of TP47 is observed only when TP47 Tnp is synthesized in cis to the DNA that contains the ends. We propose that Lys54 makes a favorable interaction with an OE-specific nucleotide pair(s), while Pro47 may cause a more favorable interaction with an IE-specific nucleotide pair(s) than it does with the corresponding OE-specific nucleotide pair(s). A model to explain the preference of TP47 Tnp for the IE in cis but not in trans is proposed.

Drosophila P-element transposase is a novel site-specific endonuclease.

We developed in vitro assays to study the first step of the P-element transposition reaction: donor DNA cleavage. We found that P-element transposase required both 5' and 3' P-element termini for efficient DNA cleavage to occur, suggesting that a synaptic complex forms prior to cleavage. Transposase made a staggered cleavage at the P-element termini that is novel for all known site-specific endonucleases: the 3' cleavage site is at the end of the P-element, whereas the 5' cleavage site is 17 bp within the P-element 31-bp inverted repeats. The P-element termini were protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable protein complex remains bound to the element termini after cleavage. These data are consistent with a cut-and-paste mechanism for P-element transposition and may explain why P elements predominantly excise imprecisely in vivo.

Sequence specificity and biochemical characterization of the RusA Holliday junction resolvase of Escherichia coli.

The RusA protein of Escherichia coli is an endonuclease that resolves Holliday intermediates in recombination and DNA repair. Analysis of its subunit structure revealed that the native protein is a dimer. Its resolution activity was investigated using synthetic X-junctions with homologous cores. Resolution occurs by dual strand incision predominantly 5' of CC dinucleotides located symmetrically. A junction lacking homology is not resolved. The efficiency of resolution is related inversely to the number of base pairs in the homologous core, which suggests that branch migration is rate-limiting. Inhibition of resolution at high ratios of protein to DNA suggests that binding of RusA may immobilize the junction point at non-cleavable sites. Resolution is stimulated by alkaline pH and by Mn2+. The protein is unstable in the absence of substrate DNA and loses approximately 80% of its activity within 1 min under standard reaction conditions. DNA binding stabilizes the activity. Junction resolution is inhibited in the presence of RuvA. This observation probably explains why RusA is unable to promote efficient recombination and DNA repair in ruvA+ strains unless it is expressed at a high level.

DNA binding by the Xis protein of the conjugative transposon Tn916.

We purified the Xis protein of the conjugative transposon Tn916 and showed by nuclease protection experiments that Xis bound specifically to sites close to each end of Tn916. These specific binding sites are close to, and in the same relative orientation to, binding sites for the N-terminal domain of Tn916 integrase protein. These results suggest that Xis is involved in the formation of nucleoprotein structures at the ends of Tn916 that help to correctly align the ends so that excision can occur.

Molecular cloning, nucleotide sequence, and function of a site-specific recombinase encoded in the major 'pathogenicity island' of Salmonella typhi.

The genome of the typhoid fever bacterium, Salmonella typhi, contains at least three large insertions ('pathogenicity islands') relative to the chromosome of Salmonella typhimurium (which is normally non-invasive for humans) [Liu, S.-L., Sanderson, K.E., 1995. Rearrangements in the genome of the bacterium Salmonella typhi. Proc. Natl. Acad. Sci. USA 92, 1018-1022]. DNA encoding a site-specific recombinase (the rci gene) and an adjacent putative pilus-tip adhesin protein (the pilV gene) was located (near min 94) in the major 'pathogenicity island' of the S. typhi chromosome, cloned, and sequenced. It was shown that the Rci protein inverted a DNA segment of 490 bp, between two 19-bp inverted repeat elements, to place either of two possible C-termini on a constant N-terminal region of the PilV protein. Both possible PilV proteins were seen when the alternative pilV genes were transcribed from the T7 promoter and translated in vivo. Both the rci and the N-terminal region of the pilV gene show a high degree of homology to genes encoded by the IncI2 plasmid R721 and the IncI1 plasmid R64. One of the possible pilV C-termini (in the pilV1 gene) is highly homologous to shufflon C (one of the possible PilV C-termini) of R64; the other possible pilV C-terminus (in the pilV2 gene) shows no homology to any published shufflon. In the R64 plasmid, the PilV proteins are pilus-tip adhesins; different PilV proteins recognize different potential recipient bacterial strains as a prelude to mating in liquid culture [Komano, T., Kim, S.-R., Yoshida, T., Nisioka, T., 1994. DNA rearrangement of the shufflon determines recipient specificity in liquid mating of IncI1 plasmid R64. J. Mol. Biol. 243, 6-9]. It is likely that S. typhi encodes pili bearing two alternative PilV proteins as tip adhesins, one of which recognizes, specifically, a membrane component of Escherichia coli K-12, while the specificity of the other PilV protein is not known.

The chloroplast-located homolog of bacterial DNA recombinase.

The cDNA for the chloroplast-located homolog of bacterial RecA protein, designated recA-AT, was placed in a plasmid appropriate for in vitro transcription and translation. Translation with 35S-labeled Met permitted demonstration of uptake of the protein product into isolated pea chloroplasts, and processing to a mature size. Preliminary evidence for the first amino acid was estimated from results using both 35S-Met and 3H-Leu for in vitro transcription and translation, followed by uptake into chloroplasts and processing. The labeled protein was subject to sequential amino acid hydrolyses, and radioactivity was measured in each round. Induction of gene transcription in leaves infiltrated with the DNA-damaging agent, methyl methane-sulfonate was shown by Northern blot analysis. Further constructs were made for over-expression of the gene in E. coli; and one out of many tried permitted production of some soluble protein. Extracts from transformed bacteria were shown to have RecA activity using the "POM" assay [Bertrand et al. (1993) Nucl. Acids Res. 21:3653] for DNA strand transfer. The protein was purified to close to homogeneity using methods developed for E. coli RecA isolation.

Specific nicking at the 3' ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP.

BACKGROUND: Tn3, a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition. The transposition of Tn3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini-Tn3 as the donor and another plasmid as the target. Transposase has the ability to bind to the 38-bp terminal inverted repeats (IRs) of Tn3. The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood. RESULTS: We found that nicking occurred efficiently in the cell-free system at each of the 3' ends of the IRs of mini-Tn3 in the closed circular or linear donor molecules. The nicking reaction required transposase and Mg2+, but did not require ATP, an ATP-regenerating system, dNTPs and polyvinyl alcohol, which were the requirements for the transposition reaction. By using the nicking assay employed here, transposase was purified almost to homogeneity. Gel filtration and sedimentation analyses indicate that transposase forms a dimer in a solution containing 0.5 M NaCl. The nicking activity of the purified transposase was weak and was found to be stimulated by a host factor. The nicking stimulation factor was subsequently purified and found to be ACP, an Escherichia coli acyl carrier protein. CONCLUSIONS: Nicking occurred efficiently at the 3' ends of mini-Tn3 in the reaction mixture containing transposase and ACP. ACP is known to act as a factor which modulates enzymes that are involved in several biological processes either in the acylated or unacylated form. ACP may also modulate transposase to initiate the transposition reaction with nicking at the 3' ends of Tn3.