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 skisserte protokollen beskriver kloning av gener av interesse i fanget merking vektor, generering av induserbare LAP-merket stabile cellelinjer, og rensing av LAP-tagget protein komplekser for proteomikk analyser. Med hensyn til andre LAP / TAP-tagging tilnærminger, har denne protokollen blitt strømlinjeformet for å være kompatibel med high-throughput tilnærminger for å kartlegge protein lokalisering og protein-protein interaksjoner innenfor enhver cellulær veien. Denne tilnærmingen har blitt mye brukt til funksjonell karakterisering av proteiner kritiske for cellesyklusprogresjon, mitotisk spindelenheten, spindel pol homeostase, og ciliogenesis for å nevne noen, og har hjulpet forståelsen av hvordan misregulation av disse proteinene kan føre til menneskelige sykdommer 15, 16,19,20. For eksempel, gruppen vår nylig utnyttet dette systemet for å definere den funksjon og regulering av STARD9 mitotiske kinesin (en kandidat kreft mål) i spindelenheten 15,21, for å definere enny molekylær koblingen mellom Tctex1d2 dynein lett kjede og korte rib polydactyly syndromer (SRPS) 19, og for å definere en ny molekylær lenke for å forstå hvordan mutasjon av Mid2 ubiquitin ligase kan føre til X-koblede utviklingshemning 16. Andre laboratorier har også med hell brukt denne metoden, inkludert en som fastslått at Tctn1, en regulator av mus pinnsvin signalering, var en del av en ciliopathy-assosierte proteinkompleks som regulerte ciliær membransammensetning og ciliogenesis i et vev-avhengig måte 22,23. Derfor kan denne protokollen grovt brukes til disseksjon av enhver cellulær veien.
Et kritisk punkt i denne protokollen er utvalget av LAP-merket stabile cellelinjer som er Hygromycin motstandsdyktig. Spesiell forsiktighet bør tas for å sikre at alle cellene i styreplate er døde før du velger foci i den eksperimentelle plate for forsterkning. Hygromycin kan også Added under rutinemessig celledyrking av LAP-merket stabile cellelinjer for å ytterligere sikre at alle cellene opprettholde LAP-merket genet av interesse på FRT nettstedet. Vi advarer at ikke alle LAP-merkede proteiner vil være funksjonelle, og at det er viktig å ha analyser på plass som kan brukes til å teste protein funksjon. Eksempler på analyser brukes til å teste protein funksjon inkluderer redning av siRNA-indusert fenotyper og in vitro aktivitetsanalyser. For å løse eventuelle problemer med tillegg av en stor LAP-tag, har vi tidligere generert TAP-tag vektorer som er kompatible med dette systemet som inneholder mindre koder, som flagg, som er mindre sannsynlighet for å hemme funksjon og lokalisering av protein av interesse 4. I tillegg LAP-tagging vektorer eksisterer for å generere C-terminal LAP-merkede proteiner eller C-terminal TAP-merkede proteiner som er kompatible med dette systemet, som kan brukes i tilfeller der en LAP / TAP tag ikke tolereres på N -terminus av et protein. I tillegg the salt og vaskemiddel konsentrasjoner av rense buffere (LAPX N) kan endres for å øke eller redusere rensing stringens hvis ingen eller for mange interaksjoner er observert. Tilsvarende er det tandem affinitetsrensing prosedyre strengere enn ett renseprosedyrer og svake interactors kan gå tapt, og således kan anvendes en enkelt rense ordning ved få eller ingen interactors er identifisert.
Det er viktig å merke seg at andre GFP epitop tagging tilnærminger finnes som tillater stor skala GFP protein merking for protein lokalisering og rensing studier 24,25. Disse inkluderer BAC TransgenOmics tilnærming som utnytter bakterie kunstige kromosomer til å uttrykke GFP-merket gener av interesse fra sitt opprinnelige miljø som inneholder alle de regulatoriske elementer, som etterligner endogene genuttrykk 24. Mer nylig har CAS9 / single-styrt RNA (sgRNA) ribonucleoprotein komplekser (RNPs) blitt brukt til å endeogenously merke gener av interesse med en split-GFP system som gjør at uttrykket av GFP-merket gener fra deres endogene genomisk loci 25. Selv om begge disse tilnærmingene aktivere uttrykk for merkede proteiner etter endogene forhold, sammenlignet med LAP-tagging protokollen beskrevet her, de tillater ikke for induserbar og fleksibel uttrykk for de merkede gener av interesse. I tillegg har de ennå å bli brukt på tandem epitop merking for TAP. Det er også viktig å merke seg at andre merkingssystemer kan også bli modifisert til å bli kompatibelt med systemet som er beskrevet her for generering av induserbare epitop-tagget stabile cellelinjer. For eksempel har nærhet avhengig biotin identifikasjon (BioID) fått stor oppmerksomhet på grunn av sin evne til å definere romlige og tidsmessige relasjoner mellom samspill proteiner 26. Denne teknikken utnytter proteinfusjoner til en promiskuøs stamme av Escherichia coli biotin-ligase BIRA, som biotinylerteer ethvert protein innen et ~ 10 nm radius av enzymet. De biotinylerte proteinene blir deretter affinitetsrenset ved å bruke biotin-affinitet fangst og analysert med henblikk på sammensetning ved hjelp av massespektrometri. BIRA vil biotinylere hvilket som helst protein i umiddelbar nærhet, selv forbigående, noe som gjør den spesielt egnet for detektering av svake vekselvirker partnere i et komplekst 27. I tillegg gjør rensing ordningen ikke nødvendiggjør at endogene protein-protein interaksjoner forbli intakt og kan utføres under denaturerende betingelser, og dermed redusere frekvensen av falske positiver. Innenfor vår nåværende protokoll, kan substitusjon av pGLAP1 vektor med en Bira merking vektortransformere dette systemet fra å identifisere protein-protein interaksjoner basert på tilhørighet til å oppdage dem basert på nærhet. Et slikt system ville være meget fordelaktig for å detektere transiente protein interaksjoner som er tilfellet mellom mange enzym-substrat-vekselvirkninger og for kartlegging av tid og rom-protein-protein interactions innenfor definerte strukturer som har blitt utført for sentrosomen og flimmerhårene 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 |