Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites

Abstract

Background: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors.

Results: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased.

Conclusion: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

Figure 1

Description of vectors for the Creator and Creator Splice systems. A) Schematic of the donor vector illustrating the essential features of the donor vector. ORFs are cloned into the Multiple Cloning Site (MCS) that is flanked by two loxP sites. The CmR ORF following the MCS, but still within the two loxP sites, ensures that both the ORF cassette from the donor vector and the acceptor vector backbone (containing the CmR promoter) are retained in the final product. The ampicillin resistance gene (AmpR) provides positive selection and the SacB gene imparts negative selection (against the donor backbone). Sequences of the novel MCS for the modified Creator and Creator Splice vectors are indicated below the diagram. The MCS starts at the AscI site and ends at the PacI site. The last four nucleotides of the loxP site are shown for orientation. The splice acceptor sequence in the Creator Splice vector is shown in green to emphasize the difference between the two vectors. B) A schematic of the modular nature of the acceptor vectors. The types of cassettes (promoter, tags, selectable markers) that can be interchanged to create vectors for specific proteomics needs are illustrated, not all combinations are available (Table 2). Abbreviations: AmpR – ampicillin resistance gene, b – bacteria expression, CmR – chloramphenicol resistance ORF, i – insect expression, m – mammalian expression, MCS – multiple cloning site, SA – splice acceptor.

Figure 2

The Mito tracking peptide targets proteins to the mitochondria. NIH 3T3 cells were transiently transfected with vectors expressing either the N-terminus of Angiomotin fused to a C-terminal ECFP (top panel) or the same protein fused to a C-terminal ECFP plus a mitochondrial tracking peptide (ECFP-mito) (bottom panel). The cells were fixed and stained with anti-ECFP (green colour in merge) that recognizes the fusion construct or anti-Cytochrome C (red colour in merge) to mark the mitochondrial membrane. DNA is stained with Hoescht (blue colour in merge). The bottom right image on both panels is a composite of all three stainings. Note that only transfected cells will display ECFP staining. Abbreviations – mito – Mito tracker, Cyt C – Cytochrome C, ECFP – enhanced cyano fluorescent protein.

Figure 3

Schematic of a typical recombination reaction in the Creator Splice system. The SD/intron, shown in purple, is the only difference between a Creator Splice acceptor and a standard acceptor vector. A Creator Splice donor and acceptor are recombined in the presence of Cre recombinase in vitro. An intron is formed in the resulting expression vector starting from the SD/intron supplied by the acceptor and ending with the splice acceptor from the donor vector. Upon transfection and transcription in mammalian cells, the intron is removed and the tag is juxtaposed onto the ORF. Abbreviations: bac – bacteria promoter, CmR – Chloramphenicol resistance ORF, kan/neoR – kanamycin and neomycin resistance gene, P – promoter, SA – splice acceptor, SD – splice donor.

Figure 4

Protein expression levels in the Creator and Creator Splice system. The indicated constructs were transiently transfected into HEK293T cells. After 48 hours, the cells were lysed and the lysate subjected to immunoblot analysis. A) WWP2 and BulI fused to Double-Myc epitope tags were detected by Myc antibody 9E10. B) ArhGEF9 and ArhGEF1 fused to Triple-Flag tags were detected by Flag antibody M2. Abbreviations: 5' SA – Vector created by recombining a Creator Splice donor vector, (splice acceptor at 5' end – V37-based), with a 5' tag acceptor vector lacking a splice donor/intron (V179). No splicing occurs in these transcripts, 5' – Vector created by recombining a donor lacking a 5' splice acceptor (V7 or V308-based) and a 5' tag acceptor vector lacking a splice donor/intron (V516- Double Myc, V179 – Triple Flag). No splicing occurs in these transcripts, SPLICE – Expression vector created by recombining a Creator Splice donor (5' splice acceptor – V37-based) and a Creator Splice 5' tag acceptor vector containing a splice donor/intron (V517 – Double Myc, V180 – Triple Flag). Resulting transcripts undergo splicing at the 5' end to remove the loxP sequence and juxtapose the tag onto the ORF, 3' – Vector created by recombining a donor lacking a 5' splice acceptor (V7 or V308-based) with a 3' tag acceptor vector lacking a splice donor/intron (V518 – Double Myc, V181 – Triple Flag). Splicing only occurs at the 3' end to remove the CmR gene (The donor contains a splice donor preceding the CmR ORF and the acceptor vector contains a splice acceptor preceding the tag), 3' SA – Vector created by recombining a Creator Splice donor vector containing a 5' splice acceptor (V37-based) and a 3' tag acceptor vector lacking a splice donor/intron (V181 -Triple Flag). Splicing only occurs at the 3' end to remove the CmR gene (same as 3' tag) and TOPO – 5' tag, non-Creator system.

Figure 5

A) Anti-Flag immunoblot of ArhGap24 with tags in the indicated positions. The transfections and expression analyses were performed in duplicates (a and b). B) Immunoblot analysis of Nadrin with tags in the indicated position. The top panel was probed with anti-Flag antibody. The immunoblot was then stripped of antibody and reprobed with anti-Nadrin (bottom panel). C) Quantitative analysis of protein levels for six different proteins with Triple-Flag epitope tags at the indicated positions. For each replicate, the band intensity, as detected by Flag M2 antibody, was normalized by the transfection efficiency as detected by fluorescent values of a co-transfecting plasmid pLP ECFP C1. The normalized band intensity was then average between the two replicates. Ratios of each tagged-protein relative to the protein expressed from the same ORF from the TOPO vector are shown above the bars. Abbreviations: SPLICE – Creator Splice Donor (V37-based) and Creator Splice 5' acceptor (V180) – splicing occurs at 5' end, 5' – standard Clontech system (donor – V7-based and acceptor – V179) – no splicing at either end, 3' – standard Clontech system (donor – V7-based and acceptor – V181) – splicing at 3' end to remove the CmR gene, 5' SA, Creator Splice Donor (V37-based) and 5' acceptor vector (V179) – no splicing at either end, TOPO – 5' tag non-recombination system, CNT – counts, RFU – relative fluorescent units.