Immunology and Infection
Published: August 12th, 2016
Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.
Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.
Trypanosoma brucei is a protozoan parasite that causes human African trypanosomasis and nagana in cattle. T. brucei is an ideal organism for the analysis of protein function due to the combination of a high quality genome, numerous proteomics and transcriptomics datasets and well developed molecular tools 1-3. Advances in proteomics and sequencing have resulted in large datasets that highlight potentially interesting genes 4-6; however, many genes have minimal information associated with them in the existing databases. There is therefore a need for a high-throughput method to aid protein functional characterization.
Expression of a tagged protein can give a multiplicity of insights into a protein's function. For example, a protein tagged with a fluorescent protein or epitope can be localized by fluorescence microscopy, which gives information about where the protein might be exerting its biological effect. Alternatively, a protein tagged with a TAP 7, HaloTag 8 or His tag can be purified for biochemical assays and identification of its interaction partners.
We recently developed a robust tagging methodology for T. brucei 9. This used long primer PCR to generate the DNA for transfection and allowed the tagging of dozens of proteins in parallel – a major improvement to existing protocols. We have now improved the scalability of this protocol in procyclic forms by an order of magnitude. Here, we present our method where we perform the PCR and transfection into 96-well plates, with the recovery and selection occurring in 24-well plates. As hundreds of proteins can now be tagged in parallel this method provides a cost-effective and feasible method for tagging the entire trypanosome genome.
1. 96-well Long Primer PCR
2. Validation of 96-well Long Primer PCR
3. 96-well Transfection
In this representative transfection, the primers were designed using the TagIt perl script 9 and synthesized commercially. The 96-well PCR was performed and validated as described (Figure 1A); in this example, 95/96 PCRs were successful (Figure 1B). In our experience, repeating failed reactions either with the original primers or re-synthesized primers does not result in a successful PCR.
The amplicons were transferred into 96-well electroporation plates for electroporation (Figure 2A) and then transferred to 24-well culture plates for recovery and selection. After sufficient time to allow the selection to occur, the wells were scored for survival prior to further analysis. In this example, 88/96 wells (92%) were positive after 15 days selection.
Figure 1: Validation of long primer PCR. (A) Pattern for transferring PCR products from 96-well PCR plate to the agarose gel for validation of PCR. (B) A representative 96-well PCR validation gel for tagging 95 genes on the N terminus, and 1 amplicon containing no 5' targeting homology to act as a negative control in the transfection. The samples are loaded onto a 1% (w/v) agarose gel and resolved at 100 V for 30 min. Please click here to view a larger version of this figure.
Figure 2: 96-well electroporation and selection. (A) Pattern for transferring electroporated cells from 96-well electroporation plate to 24-well tissue culture plates. (B) A schematic showing a typical live/dead scoring after 15 days of selection in 24-well plates. Green represents a successful transfection, grey represents a well with only dead cells. In this example, 88/96 (92%) of wells contained successfully transfected parasites. Please click here to view a larger version of this figure.
Dramatic improvements in the sensitivity of proteomics and transcriptomics methods in the last 5-10 years has provided valuable data on thousands of genes and their products. However, the tools to address the function of these proteins have not kept pace.
Tagging a protein facilitates numerous experiments to determine its function. For example, a protein can be fused to a fluorescent protein in a variety of different colors to facilitate localization and co-localization studies. Tags developed for electron microscopy, such as APEX2 or miniSOG 11,12, allow ultrastructural localization of the tagged protein. Tags for biochemistry, such as the TAP tag, and ProtC-TEV-ProtA (PTP) tag 7,13 allow purification of complexes associated with the protein for identification of binding partners or in vitro biochemical assays.
The specific steps that are critical to the success of the protocol are: the incorporation of DMSO into the PCR Master Mix 1, freezing of the PCR Master Mix 1 prior to the addition of the Master Mix 2, the use of the commercial polymerase in Master Mix 2 and the modification of the cytomix electroporation buffer. In our experience, it is necessary to use double the number of cells for C terminal tagging transfections as for N terminal tagging transfections in order to achieve a similar proportion of positive wells. Therefore, all steps should be performed as described.
This technique is only likely to be successful when transfecting the insect procyclic form trypanosome. Bloodstream forms have a lower transfection efficiency 14, moreover they are likely to die during selection due to density-dependent toxicity that is unrelated to the selective drug. Therefore, our previous protocol represents the current best technology for tagging of bloodstream form trypanosomes 9. It is also likely that the transfection efficiency will vary dependent upon the specific trypanosome isolate. This protocol was optimized using 927 SMOX procyclic forms – other strains may require additional optimization. Measures that may increase the probability of success include: increasing the amount of PCR amplicon, increasing the number of cells included in the transfection.
We present a method where hundreds of proteins can be tagged in parallel. This will facilitate large scale studies on localization and interaction, complementing existing large datasets and providing invaluable information to the community. Primer templates are available upon request and a list of plasmids templates is available from: http://www.sdeanresearch.com/
The authors have nothing to disclose.
SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.
|desalt only (do not use additional purification)
|Included with the Expand HiFi pack
|Must be PCR grade
|Expand HiFi polymerase
|HT-200 plate handler
|Handles the 96-well eletroplates
|MOS 96 ELECTROPLATE 4mm
|Electroplate for the 96-well electroporation
|Blasticidin S Hydrochloride
|Hygromycin b Gold
|225cm TC Flask, canted neck, phenolic cap
|24 well culture plates
|Eppendorf® PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes
|96-well Multiply® PCR plate with lateral skirt
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