Serine integrase chimeras with activity in E. coli and HeLa cells

Abstract

In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{-1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

Keywords: Chimeric recombinase; Genome engineering; Hybrid protein; Phage integrase; Sequence-specific recombination.

Fig. 1.. Histogram of serine recombinase lengths.

All proteins with an InterPro (Hunter et al., 2012) serine recombinase catalytic domain (IPR006119; 35,076 entries) were clustered (13,019 clusters). The mean protein length of each cluster was computed, and the distribution of these lengths is presented here as a histogram. The list of putative serine recombinases was assembled with a custom script that scanned the entire InterPro “Protein matched complete” XML flatfile (∼75 GiB uncompressed; downloaded on Feb. 14, 2014) for proteins with an IPR006119 domain (35,076 proteins found). Protein sequences were downloaded from UniProt (UniProt Consortium, 2012) and were validated via CRC64 checksum comparison with InterPro. CD-HIT (Li and Godzik, 2006) version 4.6.1 was used to perform the clustering with the following parameters: 95% identity cutoff, 95% size cutoff, five character word size. Because the smallest characterized serine integrases (A118 and U153, accession numbers Q9T193 and Q8LTD8, respectively) are both 452 residues in length, we estimate that there are at least 4,000 unique putative large serine recombinases in the InterPro database as of February 14, 2014.

Fig. 2.. Hybrid integrases: architectures and naming.

(A) Domain organization of parental and chimeric integrases. Pairwise phiBT1-phiC31 and TG1-phiC31 integrase domain alignments were performed, and the percent similarity is shown between the respective regions. EMBOSS Needle with the BLOSUM62 scoring matrix was used for all sequence alignments (Rice et al., 2000). The architectures of two representative hybrids are displayed below the parental enzymes. All chimeric integrases described in this study have been assigned systematic names that specify the parental proteins and fusion indices used to create them. (B) Detailed view of the relative indexing scheme that we developed to specify protein fusions. See the text for an explanation of our naming system.

Fig. 3.. Chimeric att-sites: organization and nomenclature.

(A) Overview of parental and hybrid att-site structure. Serine integrase att-sites consist of a dinucleotide core (black bar) that is flanked by two half-sites. Checkered patterns are used here to indicate attB half-sites; solid colors are used for attP. Drawings are not to scale. See the text for an explanation of our hybrid att-site naming system. (B) Detailed overview of parental and hybrid attB seqences. Mismatches in the B0–B3 and B0–B6 alignments are underlined. CbC B0 and TcT B0 are equivalent to the phiC31 and TG1 attB sites, respectively. (C) Detailed overview of parental and hybrid attP seqences. Mismatches in the P0–P3 and P0–P6 alignments are underlined. CbC P0 and TcT P0 are equivalent to the phiC31 and TG1 attP sites, respectively. In (B) and (C), only a central 26 nucleotide window is shown; see supplementary material Table S2 for the full sequences of all att-sites used in our study.

Fig. 4.. E. coli activity assay and results.

(A) Simplified diagram of detection scheme for integrase activity. In the un-flipped state, the lacZα fragment is not expressed, as its ORF is inverted relative to the upstream promoter. Expression of lacZα occurs when the flanking att-sites are recombined. (B) To detect active integrases, we grew transformed E. coli on plates with X-gal to detect alpha-complementation of beta-galactosidase. (C) Summary of results for recombination attempts with the CT{8,8} hybrid and TcT att-sites. See the text for an explanation of hybrid att-site nomenclature. Active and inactive att-site pairings are indicated with ‘+’ and ‘−’, respectively. TG1 integrase is able to recombine pairings with an inset “T”. No recombination of the TcT B6 hybrid site was attempted with TG1 integrase. (D) Results for recombination of CbC att-site pairings with BC{−1} hybrid. Wild-type phiC31 int is able to recombine pairings with an inset “C”, but not those marked with “X”. No recombination of the CbC B6 hybrid site was attempted with phiC31 integrase. Results from one of three independent representative trials was used to construct each result summary.

Fig. 5.. HeLa activity assay and results.

(A) EGFP inversion test for integrase expression vector activity. In the starting substrate plasmid, EGFP is not significantly expressed because its ORF is inverted relative to the upstream promoter. If the flanking att-sites are recombined by an active integrase, EGFP expression is triggered. (B) Normalized integrase expression vector inversion efficiency. HeLa cells were transfected with different combinations of a protein expression plasmid and an EGFP inversion vector. The abbreviations “−”, “C31”, “BC”, and “BT1” refer to the negative-control, phiC31, BC{−1} and phiBT1 integrase expression plasmids, respectively. “P0”, “P3”, “B0” and “B3” all refer to the respective CbC att-sites. To calculate the normalized efficiency for each plasmid combination, we divided the percentage of EGFP-positive cells by the mean transfection efficiency of the positive control inversion plasmid (“LR”, pMF-CLCR; supplementary material Fig. S1E). In all trials, each plasmid combination was transfected in triplicate. The error bars indicate the standard error of the mean. Data from one of three independent representative trials is shown here.

Fig. 6.. HeLa pseudo site integration assay.

(A–C) Maps of pDB2, pDCP and pDCbC-P3 donor plasmids. (D) Mean counts of G418-resistant colonies. HeLa cells were transfected with different combinations of an integrase expression vector and a donor plasmid, and were then subjected to G418 selection. The abbreviations “−”, “C31” and “BC” refer to the negative control, phiC31 and BC{−1} integrase expression plasmids, respectively. The pDB2, pDCP and pDCbC-P3 vectors carry a 285 bp phiC31 attB, 50 bp phiC31 attP and 50 bp CbC P3 site, respectively. In all trials, each plasmid combination was transfected in triplicate. The error bars indicate standard error of the mean. Data from one of two independent representative trials is shown here.

Fig. 7.. Summary of hybrid integrase activity results.

(A) Hybrid activity in E. coli. Chimeric integrases have been grouped according to their activity and type. Active hybrids were able to perform recombination on at least one of the indicated att-site pairings, while inactive hybrids were not. Positive recombination results are indicated with a checkmark and negative results are marked with an “x”. Untested combinations are denoted with a dash (“−”) and pairings that produced weak positive results (light blue colonies) are marked with a tilde (“∼”). Hybrids that aggregated when overexpressed in E. coli were not subjected to any recombination assays. (B) Hybrid activity in HeLa. The CT{8,8} integrase TcT P3 × B3 reaction is marked with a tilde because the recombination product could only be detected via PCR.