We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.
Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein’s spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and protein-protein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.
To investigate the cellular function of an uncharacterized protein it is important to determine its in vivo spatiotemporal subcellular localization and its interacting protein partners. Traditionally, single and tandem epitope tags fused to the N or C-terminus of a protein of interest have been used to facilitate protein localization and protein interaction studies. For example, the tandem affinity purification (TAP) technology has enabled the isolation of native protein complexes, even those that are in low abundance, in both yeast and mammalian cell lines1,2. The localization and affinity purification (LAP) technology, is a more recent development that modifies the TAP procedure to include a localization component through the introduction of the green fluorescent protein (GFP) as one of the epitope tags3. This approach has given researchers a deeper understanding of a protein’s subcellular localization in living cells while also retaining the ability to perform TAP complex purifications to map protein-protein interaction networks.
However, there are many issues associated with the use of TAP/LAP technologies that has hampered their widespread use in mammalian cells. For example, the length of time that is necessary to generate a stable cell line expressing a TAP/LAP tagged protein of interest; which typically relies on cloning the gene of interest into a viral vector and selecting single cell stable integrants with the desired expression level. Additionally, many cellular pathways are sensitive to constitutive protein overexpression (even at low levels) and can arrest cells or trigger cell death over time making the generation of a TAP/LAP stable cell line impossible. These and other constraints have impeded LAP/TAP methodologies from becoming high-throughput systems for protein localization and protein complex elucidation. Therefore, there has been considerable interest in the development of an inducible high-throughput LAP-tagging system for mammalian cells that takes advantage of current innovations in cloning and cell line technologies.
Here we present a protocol for generating stable cell lines with Doxycycline/Tetracycline (Dox/Tet) inducible LAP-tagged proteins of interest that applies advances in both cloning and mammalian cell line technologies. This approach streamlines the acquisition of data with regards to LAP-tagged protein subcellular localization, protein complex purification and identification of interacting proteins4. Although affinity proteomics utilizes a wide range of techniques for protein complex elucidation5, our approach is beneficial for expediting the identification of these complexes and their native interaction networks and is amenable to high-throughput protein tagging that is necessary to investigate complex biological pathways that contain a multitude of protein constituents. Key to this approach are advancements in cloning strategies that enable high fidelity and expedited cloning of target genes into an array of vectors for gene expression in vitro, in various organisms like bacteria and baculovirus, and in mammalian cells6,7. Additionally, the ORFeome collaboration has cloned thousands of sequence validated open reading frames in vectors that incorporate these advances in cloning, which are available to the scientific community8-11. In our system, the pGLAP1 LAP-tagging vector enables the simultaneous cloning of a large number of clones, which facilitates high-throughput LAP-tagging. This expedited cloning procedure is coupled to a streamlined approach for generating cell lines with LAP-tagged genes of interest inserted at a single pre-determined genomic locus. This makes use of cell lines that contain a single flippase recognition target (FRT) site within their genome, which is the site of integration for LAP-tagged genes. These cell lines also express the tetracycline repressor (TetR) that binds to Tet operators (TetO2) upstream of the LAP-tagged genes and silences their expression in the absence of Dox/Tet. This allows for Dox/Tet inducible expression of the LAP-tagged protein at any given time. Having the capability of inducible LAP-tagged protein expression is critical, since many cellular pathways are sensitive to the levels of critical proteins governing the pathway and can arrest cell growth or trigger cell death when these proteins are constitutively overexpressed, even at low levels, making the generation of non-inducible LAP-tagged stable cell lines impossible12.
Den skitserede protokol beskriver kloningen af gener af interesse ind i LAP-tagging vektor, generering af inducerbare LAP-mærkede stabile cellelinjer, og oprensningen af LAP-mærkede protein komplekser til proteomikanalyser. Med hensyn til andre LAP / TAP-tagging tilgange, er denne protokol blevet strømlinet for at være forenelig med high-throughput metoder til at kortlægge protein lokalisering og protein-protein interaktioner inden for enhver cellulær vej. Denne fremgangsmåde har været almindeligt anvendt til den funktionelle karakterisering af proteiner kritiske for cellecyklusprogression, mitosespindelen forsamling, spindel pole homeostase, og ciliogenesis at nævne nogle få, og har hjulpet forståelsen af, hvordan fejlagtig regulering af disse proteiner kan føre til sygdomme hos mennesker 15, 16,19,20. For eksempel, vores gruppe for nylig udnyttet dette system til at definere funktionen og reguleringen af STARD9 mitotiske kinesin (en kandidat kræft mål) i spindel samling 15,21, for at definere enny molekylær forbindelse mellem Tctex1d2 dynein let kæde og korte ribben polydaktyli syndromer (SRP'er) 19, og til at definere en ny molekylær link til at forstå, hvordan mutation af Mid2 ubiquitinligase kan føre til X-bundne intellektuelle handicap 16. Andre laboratorier har også med held anvendt denne metode, herunder en, der bestemmes, at Tctn1, en regulator af muse hedgehog signalering, var en del af en ciliopathy-associeret protein-kompleks, at reguleret ciliær membransammensætning og ciliogenesis i et væv-afhængig måde 22,23. Derfor kan denne protokol i store træk anvendes på dissektion af enhver cellulær vej.
Et kritisk trin i denne protokol er det udvalg af LAP-mærkede stabile cellelinier, der er hygromycin-resistent. Der skal udvises særlig omhu for at sikre, at alle celler i kontrolgruppen plade er døde, før du vælger foci i den eksperimentelle plade til forstærkning. Hygromycin kan også Added under rutinemæssig celle dyrkning af LAP-mærkede stabil cellelinjer til yderligere at sikre, at alle celler opretholde LAP-mærkede gen af interesse ved FRT-stedet. Det skal understreges, ikke alle LAP-mærkede proteiner vil være funktionel, og at det er vigtigt at have assays på plads, der kan anvendes til at teste proteinfunktion. Eksempler på anvendte assays til at teste proteinfunktion omfatter redningen af siRNA-induceret fænotyper og in vitro aktivitetsassays. For at løse eventuelle problemer med tilføjelse af et stort LAP-tag, har vi tidligere genereret TAP-tag vektorer er forenelige med dette system, der indeholder mindre tags, såsom FLAG, som er mindre tilbøjelige til at inhibere funktionen og lokalisering af proteinet af interesse fire. Endvidere findes LAP tagging vektorer til frembringelse C-terminale LAP-mærkede proteiner eller C-terminale TAP-mærkede proteiner, der er kompatible med dette system, som kan anvendes i tilfælde, hvor en LAP / TAP tag ikke tolereres ved N terminus af et protein. Derudover the salt og opvaskemiddel koncentrationer i rensningsanlægget buffere (LAPX N) kan modificeres til at øge eller mindske rensning stringens, hvis der observeres ingen eller for mange interaktioner. Tilsvarende affinitetsoprensning procedure tandem er strengere end enkelt oprensningsprocedurer og svage interaktionskandidater kan gå tabt, således en enkelt rensningsskema kan anvendes, når få eller ingen interaktionskandidater identificeres.
Det er vigtigt at bemærke, at der findes andre GFP-epitop tagging tilgange, tillader stor skala GFP protein tagging for protein lokalisering og rensning studier 24,25. Disse omfatter BAC TransgenOmics tilgang, der anvender bakterielle kunstige kromosomer til at udtrykke GFP-mærkede gener af interesse fra deres native miljø, der indeholder alle de regulatoriske elementer, som efterligner endogene genekspression 24. For nylig er CAS9 / single-styret RNA (sgRNA) ribonukleoprotein komplekser (RNP'er) blevet anvendt til at afslutteogenously tag gener af interesse med en split-GFP system, der tillader ekspression af GFP-mærkede gener fra deres endogene genomiske loci 25. Selv om begge disse tilgange muliggøre ekspressionen af mærkede proteiner under endogene betingelser, sammenlignet med den her beskrevne LAP-tagging protokol, de ikke tillader inducerbar og afstemmelige ekspression af de taggede gener af interesse. Derudover har de endnu ikke anvendes på tandem epitop tagging for TAP. Det er også vigtigt at bemærke, at andre tagging systemer også kan modificeres til at blive kompatible med systemet beskrevet her til frembringelse af inducerbare epitopmærkede stabile cellelinjer. For eksempel har nærhed-afhængig biotin identifikation (BioID) vundet stor opmærksomhed på grund af sin evne til at definere rumlige og tidsmæssige relationer blandt interagerende proteiner 26. Denne teknik udnytter proteinfusioner til en promiskuøs stamme af Escherichia coli biotinligase BirA, som biotinyleres helst protein i en ~ 10 nm radius af enzymet. De biotinylerede proteiner er derefter affinitetsoprenset under anvendelse af biotin-affinitetsindfangning og analyseret for sammensætning ved massespektrometri. BirA vil biotinylere helst protein i umiddelbar nærhed, selv kortvarigt, hvilket gør det særligt velegnet til detektering svagere interagerende partnere i et kompleks 27. Derudover betyder rensning ordningen ikke nødvendiggør, at endogene protein-protein interaktioner forblive intakt og kan udføres under denatureringsbetingelser, og dermed reducere antallet af falske positiver. Inden for vores nuværende protokol, kan substitution af pGLAP1 vektor af en BirA-tagging vektor omdanne dette system fra at identificere protein-protein interaktioner baseret på affinitet til at opdage dem baseret på nærhed. Et sådant system ville være yderst fordelagtig til detektion af forbigående protein-interaktioner som det er tilfældet mellem mange enzym-substrat-interaktioner og til kortlægning af det Spatiotemporal protein-protein-interactions inden definerede strukturer som det er blevet udført for centrosom og cilier 26,28.
The authors have nothing to disclose.
This work was supported by a National Science Foundation Grant NSF-MCB1243645 (JZT), any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Flp-In T-REx Core Kit | Invitrogen | K6500-01 | Kit for generating cell lines that contain an FRT site and TrtR expression |
PETG, 5X | Nunc, Inc. | 73520-734 | Roller bottle for growing cells |
PETG, 2.5X | Nunc, Inc. | 73520-420 | Roller bottle for growing cells |
Cell stackers | Corning CellSTACK | 3271 | Cell stacker for growing cells |
500 mL conical centrifuge tubes | Corning | 431123 | Tubes for harvesting cells |
Anti-GFP antibody | Invitrogen | A11122 | Rabbit anti GFP antibody |
Affiprep Protein A beads | Biorad | 156-0006 | Used as a matrix for conjugating anti-GFP antibodies |
Dimethylpimelimidate (DMP) | ThermoFisher Scientific | 21667 | Used for conjugating anti-GFP antibodies to Protein A beads |
TLA100.3 tubes | Beckman | 349622 | Tubes for centrifuging protein lysates during the clearing step |
TEV protease | Invitrogen | 12575-015 | Used for cleaving the GFP tag off of N-terminal LAP-tagged proteins |
Precession Protease | GE Healthcare | 27-0843-01 | Used for cleaving the GFP tag off of C-terminal LAP-tagged proteins |
S-protein agarose | Novagen | 69704 | Used as a second affinity matrix during the purification of LAP-tagged protein complexes |
QIAquick DNA gel extraction kit | Qiagen | 28704/28706 | For use in purifying PCR products from an agarose gel |
BP clonase II | Invitrogen | 11789020 | Used for cloning ORF PCR products into the pDONR221 shuttle vector |
LR clonase II | Invitrogen | 11791020 | Used for cloning the ORF of the gene of interest into the pGLAP1 LAP-tagging vector |
ccdB Survival 2 T1R E. coli | Invitrogen | A10460 | Used for propgating shuttle vectors and pGLAP empty vectors |
Fugene 6 | Promega | E2691 | Transfection reagent for transfecting vectors into human cells |
Tetracycline | Invitrogen | Q100-19 | Drug for inducing Dox/Tet inducible protein expression |
Doxycycline | Clontech | 631311 | Drug for inducing Dox/Tet inducible protein expression |
Hygromycin B | Invitrogen | 10687010 | Drug for selecting stable LAP-tagged integrants |
Kanamycin | Corning | 61-176-RG | Drug for selecting Kanamycin resistant bacterial colonies |
Ampicillin | Fisher | BP1760-5 | Drug for selecting Ampicillin resistant bacterial colonies |
4-20% Tris Glycine SDS-PAGE gels | Biorad | 4561094 | Used for separating protein samples and final LAP-tag purification eluates |
Silver Stain Plus Kit | Biorad | 1610449 | Used for silver staining the eluates of LAP-tagged pufications and samples collected throughout the purification process |
Coomassie Blue stain | Invitrogen | LC6060 | Used for staining SDS-PAGE gels to visulize LAP-tagged purifications and cutting out protein bands, mass spectrometry compatible |
Shuttle vector pDONR221 | Invitrogen | 12536017 | Shuttle vector for cloning the ORFs of genes of interest |
Flippase expressing vector pOG44 | Invitrogen | V600520 | Vector that expresses the Flippase recombinase for integrating LAP-tagged genes into the genome of FRT site containing cell lines |
Platinum Taq DNA Polymerase | ThermoFisher Scientific | 10966018 | Used for PCR amplification of the ORFs of genes of interest |
4X Laemmli sample buffer | Biorad | 1610747 | Sample buffer for eluting purified LAP-tagged protein complexes from the bead matrix |
Luria broth (LB) media | Fisher | BP9723-2 | Used for growing DH5α bacteria |
DNA miniprep kit | Promega | A1222 | Used for making DNA plasmid minipreps |
DMEM/F12 media | Hyclone | SH30023.01 | For growing Hek293 human cells |
FBS lacking Tet | Altanta Biologicals | S10350 | Used for making -Tet DMEM/F12 media for generating and growing inducible LAP-tagged stable cell lines |
Trypsin | Hyclone | SH30042.01 | For lifting Hek293 cell foci from plates |
Protease inhibitor tablets | Roche | 11836170001 | Used for making protocol buffers, EDTA-free |
10% nonyl phenoxypolyethoxylethanol | Roche | 11332473001 | Used for making protocol buffers |
PBS | Corning | 21-040-CM | Used for making protocol buffers |
Tween-20 | Fisher | BP337-500 | Used for making protocol buffers |
Sodium Borate | Fisher | S249-500 | Used for making protocol buffers |
Boric Acid | Fisher | A78-500 | Used for making protocol buffers |
Ethanolamine | Calbiochem | 34115 | Used for making protocol buffers |
NaCl | Fisher | P217-3 | Used for making protocol buffers |
KCl | Fisher | BP358-10 | Used for making protocol buffers |
Dithiothreitol (DTT) | Fisher | BP172-25 | Used for making protocol buffers |
MgCl2 | Fisher | M33-500 | Used for making protocol buffers |
Tris base | Fisher | BP152-5 | Used for making protocol buffers |