Preparation of cell-permeable Cre recombinase by expressed protein ligation

Background

Protein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli.

Results

Expressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC.

Conclusions

Collectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0126-z) contains supplementary material, which is available to authorized users.     

Stoichiometry of the Cre recombinase bound to the lox recombining site.

The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site. The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region. Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site. We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site.     

Directed evolution of recombinase specificity by split gene reassembly

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications.     

DNA specificity of the Cre recombinase resides in the 25 kDa carboxyl domain of the protein

The Cre protein of bacteriophage P1 is a 38.5 kDa site-specific recombinase that belongs to the Int family of recombination proteins. Cre acts by binding specifically to a 34 base-pair sequence, lox, where it carries out recombination. A limited chymotryptic digest of Cre resulted in two fragments of sizes 25 and 13.5 kDa, respectively. The sequence of the amino terminus of the purified 25 kDa peptide demonstrates that this peptide represents the carboxyl-terminal portion of the Cre protein. A truncated version of the cre gene was constructed which produces only the 25 kDa peptide. The 25 kDa peptide is capable of specific binding to the lox site, but binds at lower affinity than does wild-type Cre. Footprinting with Fe-EDTA indicates that the 25 kDa peptide protects the inverted repeats of the lox site but shows only partial protection of the spacer region. This is in contrast to the footprint obtained with wild-type Cre which protects the entire spacer region.     

Synapsis of loxP sites by Cre recombinase

Cre recombinase catalyzes site-specific recombination between 34-bp loxP sites in a variety of topological and cellular contexts. An obligatory step in the recombination reaction is the association, or synapsis, of Cre-bound loxP sites to form a tetrameric protein assembly that is competent for strand exchange. Using analytical ultracentrifugation and electrophoresis approaches, we have studied the energetics of Cre-mediated synapsis of loxP sites. We found that synapsis occurs with a high affinity (Kd = 10 nM) and is pH-dependent but does not require divalent cations. Surprisingly, the catalytically inactive Cre K201A mutant is fully competent for synapsis of loxP sites, yet the inactive Y324F and R173K mutants are defective for synapsis. These findings have allowed us to determine the first crystal structures of a pre-cleavage Cre-loxP synaptic complex in a configuration representing the starting point in the recombination pathway. When combined with a quantitative analysis of synapsis using loxP mutants, the structures explain how the central 8 bp of the loxP site are able to dictate the order of strand exchange in the Cre system.     

Genomic targeting with purified Cre recombinase.

Purified Cre recombinase protein introduced directly into cultured mammalian cells by lipofection catalyzes both site-specific chromosomal integration of a co-transfected lox targeting vector and precise excision of genomic DNA flanked by directly repeated lox sites. This procedure eliminates the need to transfect cre expression plasmids to activate recombination at lox sites. We used this simplified procedure to investigate the effect on targeting efficiency of both lox vector design and chromosomal position of the lox target. We show that such chromosomal position effects can exert at least a 50-fold per lox target difference in targeting efficiency in a human osteosarcoma cell line.     

Sequence of the loxP site determines the order of strand exchange by the Cre recombinase

Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.     

Alteration of substrate specificity of aspartase by directed evolution

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.     

Directional resolution of synthetic holliday structures by the Cre recombinase.

The Cre recombinase of bacteriophage P1 cleaves its target site, loxP, in a defined order. Recombination is initiated on one pair of strands to form a Holliday intermediate, which is then resolved by cleavage and exchange of the other pair of strands to yield recombinant products. To investigate the influence of the loxP sequence on the directionality of resolution, we constructed synthetic Holliday (chi) structures containing either wild-type or mutant lox sites. We found that Cre preferentially resolved the synthetic wild-type chi structures on a particular pair of strands. The bias in the direction of resolution was dictated by the asymmetric loxP sequence since the resolution bias was abolished with symmetric lox sites. Systematic substitutions of the loxP site revealed that the bases immediately 5' to the scissile phosphodiester bonds were primarily responsible for the directionality of resolution. Interchanging these base pairs was sufficient to reverse the resolution bias. The Cre-lox co-crystal structures show that Lys(86) makes a base-specific contact with guanine immediately 5' to one of the scissile phosphates. Substituting Lys(86) with alanine resulted in a reduction of the resolution bias, indicating that this amino acid is important for establishing the bias in resolution.     

Alteration of enzyme specificity and catalysis by protein engineering.

New substrate specificities can be introduced into existing enzymes for the purpose of making them more suitable for the chemoenzymic synthesis of single compound drugs and other chiral compounds. The most productive route used in the past year has involved the utilization of the catalytic and substrate-binding properties from homologous enzymes found in nature, one example being the broadening of the substrate specificity of yeast alcohol dehydrogenase. Other highlights include the creation of thermostable dehydrogenases that will interconvert NADPH and NADH, and the design of mutant enzymes with improved catalytic rates compared with their wild-type counterparts.     

Intein-mediated rapid purification of Cre recombinase

Cre recombinase produced by bacteriophage P1 catalyzes site-specific recombination of DNA between loxP recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning. Recombinant Cre has been overproduced in Escherichia coli and its purification involves multiple steps. In this report, we used an "intein" fusion system to express Cre as a C-terminal fusion to a modified protein splicing element, i.e., intein. The modified intein contained a Bacillus circulans chitin-binding domain which allowed binding of the fusion protein on a chitin column and could be induced to undergo in vitro peptide bond cleavage which specifically released Cre from the column. Using the intein system, we have obtained highly pure nontagged Cre after just a single chromatographic step, which corresponded to approximately 80% recovery and 27-fold purification. The activity of the purified Cre was determined in an in vitro assay system and was found to remain stable over a period of more than 6 months.     

Generation of chickens expressing Cre recombinase

Cre recombinase has been extensively used for genome engineering in transgenic mice yet its use in other species has been more limited. Here we describe the generation of transgenic chickens expressing Cre recombinase. Green fluorescent protein (GFP)-positive chicken primordial germ cells were stably transfected with β-actin-Cre-recombinase using phiC31 integrase and transgenic chickens were generated. Cre recombinase activity was verified by mating Cre birds to birds carrying a floxed transgene. Floxed sequences were only excised in offspring from roosters that inherited the Cre recombinase but were excised in all offspring from hens carrying the Cre recombinase irrespective of the presence of the Cre transgene. The Cre recombinase transgenic birds were healthy and reproductively normal. The Cre and GFP genes in two of the lines were closely linked whereas the genes segregated independently in a third line. These founders allowed development of GFP-expressing and non-GFP-expressing Cre recombinase lines. These lines of birds create a myriad of opportunities to study developmentally-regulated and tissue-specific expression of transgenes in chickens.     

Retina-specific gene excision by targeted expression of Cre recombinase

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho–Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho–Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987 kb fragment (pNCHS4 Rho–NLS–cre) containing human rhodopsin promoter was cloned. The Rho–Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316 kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho–Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho–Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.     

Spermatocyte-specific gene excision by targeted expression of Cre recombinase

Transgenic mice carrying the coding sequence of the Cre recombinase, whose expression was driven by the spermatocyte-specific Pgk-2 promoter, were generated. These mice were crossed with a reporter transgenic line, which produces beta-galactosidase depending on the occurrence of loxP-mediated DNA recombination. When DNA of the offspring was analyzed by PCR and Southern blotting, signals that appear after the recombination were detectable only in the testis. Histochemical analyses revealed that beta-galactosidase was present in spermatocytes and spermatogenic cells at later differentiation stages. However, the distribution of the protein was not uniform in all spermatocytes. Analyses of genomic DNA of the next generation indicated that recombination took place in about 70% of spermatogenic cells. From these results, we concluded that this transgenic line possessing Pgk-2-driven expression of the Cre recombinase should be useful for identifying spermatogenic genes that function at or after the spermatocyte stage.     

Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis

Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed.     

Regulation of Cre recombinase activity by the synthetic steroid RU 486.

To create a strategy for inducible gene targeting we developed a Cre-lox recombination system which responds to the synthetic steroid RU 486. Several fusions between Cre recombinase and the hormone binding domain (HBD) of a mutated human progesterone receptor, which binds RU 486 but not progesterone, were constructed. When tested in transient expression assays recombination activities of all fusion proteins were responsive to RU 486, but not to the endogenous steroid progesterone. However, the observed induction of recombination activity by the synthetic steroid varied between the different fusion proteins. The fusion with the highest activity in the presence of RU 486 combined with low background activity in the absence of the steroid was tested after stable expression in fibroblast and embryonal stem (ES) cells. We could demonstrate that its recombination activity was highly dependent on RU 486. Since the RU 486 doses required to activate recombination were considerably lower than doses displaying anti-progesterone effects in mice, this system could be used as a valuable tool for inducible gene targeting.     

Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein

Bacteriophage P1 encodes a site-specific recombination system that consists of a site (loxP) at which recombination occurs and a gene, cre, whose protein product is essential for recombination. The loxP-Cre recombination event can be studied in greater detail by the use of an in vitro system that efficiently carries out recombination between two loxP sites. This paper presents a purification and characterization of the Cre protein (Mr = 35,000), which is the only protein required for the in vitro reaction. No high energy cofactors are needed. The purified Cre protein binds to loxP-containing DNA and makes complexes that are resistant to heparin. Cre efficiently converts 70% of the DNA substrate to products and appears to act stoichiometrically. The action of Cre on a loxP2 supercoiled substrate containing two directly repeated loxP sites results in product molecules that are topologically unlinked. Several models to account for the ability of Cre to produce free supercoiled products are discussed.     

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12–rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05–0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48–72 h, with an EC₅₀ of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.     

Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate

Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids.