Lentiviral Fluorescent Protein Expression Vectors for Biotinylation Proteomics Protocol

Overview

Introduction of Biotinylation Proteomics Protocol

A variety of biochemical methods exist for studying protein– protein interactions in mammalian cells, including those based on fluorescence-activated cell sorting (FACS). Strouboulis and colleagues developed a method in which a recombinant protein is tagged with a peptide that is biotinylated in vivo by the coexpressed Escherichia coli BirA biotin ligase. A strength of this approach is the very high affinity interaction between biotinylated substrates and streptavidin (Kd=10−15), which allows high stringencies to be employed during purification. The biotinylation tagging method described here has been successfully used for the single-step purification of a number of transcription factor complexes in nuclear extracts of mammalian cells.

In the initial version of the method, expression cassettes encoding the tagged protein of interest and BirA were subcloned into separate plasmids that also carried a selectable drug resistance gene, the neomycin phosphotransferase gene and the puromycin N-acetyltransferase gene, respectively. Typically, target cells would be transfected with the BirA plasmid by physical methods, such as electroporation, stable clones obtained by the selection of puromycin resistance, and then screened for BirA expression by Northern or Western blot analysis. An appropriate BirA expressing clone would then be transfected with the vector encoding the tagged protein of interest and stable cells expressing the tagged protein biotinylated by BirA obtained by selection for cells that were resistant to the neomycin analog geneticin as well as to puromycin. A drawback of this experimental design is that the optimal concentration of drugs used for the selection depends on the cell line and needs to be determined a priori. Moreover, the selection for drug resistance of the transfected plasmids does not necessarily ensure the coexpression of BirA or the tagged gene of interest.

Hoang and colleagues reported a modification of this methodology in which the tagged gene of interest was coexpressed from a lentiviral vector on a bicistronic transcript that also contained the BirA gene. In addition, the BirA gene was expressed as a fusion protein with the green fluorescent protein (BirA-GFP), allowing stably transduced cells coexpressing the tagged gene of interest and BirA to be isolated by FACS. As described below, we have further modified the FACS-based strategy of Hoang and colleagues. The expression system we have developed consists of two components: a lentiviral vector coexpressing the gene of interest containing a biotinylation tag together with a downstream yellow fluorescent protein (YFP) gene via an encephalomyocarditis virus internal ribosome entry site (IRES); and a second lentiviral vector expressing GFP-BirA.

Our work is focused on TLX1 (T-cell leukemia homeobox 1, previously known as HOX11 or TCL3), an evolutionarily conserved member of the dispersed NKL (NK-Like or NK-Linked) subclass of homeobox genes. The murine ortholog of human TLX1 is essential for splenogenesis and required for the development of certain neurons. Although TLX1 is not expressed in the hematopoietic system, its inappropriate activation is a recurrent event in human T cell acute lymphoblastic leukemia (T-ALL). The manner in which deregulated TLX1expression induces neoplastic conversion remains to be fully elucidated. Several lines of evidence indicate that TLX1 functions as a transcriptional regulator that can either activate or repress gene expression via direct or indirect modes of action. In this regard, TLX1 has been reported to form protein– protein interactions with other transcription factors as well as with a number of transcriptional coregulators and chromatin-modifying enzymes. Among the molecules that have been identified are: CTF1, a ubiquitous transcription factor that associates with TFIIB and the basal transcription machinery; MEIS and PBX members of the TALE (three amino acid loop extension) superclass of homeodomain proteins; the acetyltransferase coactivator CREB-binding protein; the serine/threonine phosphatases PP1 and PP2A; and the eukaryotic initiation factor 4E (eIF4E).

Figure 1. FACS-based binary in vivo biotinylation tagging system.

We recently implemented the in vivo biotinylation tagging approach to isolate and characterize the various TLX1 protein complexes in T-ALL cells. Our initial attempts to coexpress biotinylation-tagged TLX1 and GFP-BirA from a bicistronic lentiviral vector were unsuccessful because the cells did not tolerate high levels of TLX1. Since BirA was expressed on the same transcript, it was selected against. As a result, much higher expression levels of BirA were achieved with the empty GFP-BirA vector, making it difficult to obtain similar levels of endogenous biotinylated proteins for comparisons between the experimental and control samples. To circumvent these problems, we designed a two-component expression system using the simian immunodeficiency virus (SIV) lentiviral vector backbone pCL20cSLFR MSCV-GFP. In pCL20cSLFR MSCV-GFP, which was constructed from the nonpathogenic SIVmac1A11 isolate, the GFP gene is expressed from an internal promoter derived from the long terminal repeat (LTR) of the murine stem cell virus (MSCV), which is highly active in most mammalian cell types. A biotinylation tagging vector expressing a COOH-terminal tagged TLX1 (TLX1bio) under the control of the MSCV LTR was created by inserting the TLX1 coding region in frame upstream of the coding sequences for the BirA target peptide linked to an IRES-YFP cassette, generating pCL20cSLFR MSCV-TLX1bio-IRES-YFP (component 1). A BirA expression vector was similarly constructed by replacing the GFP gene of pCL20cSLFR MSCV-GFP with GFP-BirA, generating pCL20cSLFR MSCV-GFP-BirA (component 2).

In pilot studies, SupT1 cells, which are negative for TLX1 expression but which are arrested at the same stage of T-cell differentiation as TLX1+ T-ALL cells, were transduced with recombinant CL20cSLFR MSCV-TLX1bio-IRES-YFP lentiviral vector particles and/or recombinant CL20cSLFR MSCV-GFP-BirA lentiviral vector particles and stable cell lines obtained by sorting for YFP and/or GFP fluorescence. SupT1 cells expressing GFP-BirA alone served as control for the binding of any biotinylated endogenous proteins to the streptavidin beads. Using single-step affinity capture on streptavidin beads, followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, we identified the Groucho/transducin-like Enhancer of split (Gro/TLE) family member TLE1 as an in vivo binding partner of TLX1. Gro/TLE proteins are regulated by multiple signaling cascades and serve as corepressors for different families of transcription factors. The transcription factors that interact with Gro/TLE corepressors contain short peptide sequences related to either WRPW or to FXIXXIL (where X is any amino acid), the latter referred to as the Engrailed homology 1 (Eh1) motif, a repression domain first identified in the Drosophila Engrailed homeodomain protein. We demonstrated that TLX1 interacts with TLE1 in vitro and in vivo through a seven amino acid sequence encompassing amino acids 19–25 (FGIDQIL) that exhibits similarity to an Eh1 motif. Moreover, we found that this motif was required for optimal regulation of expression of two TLX1 target genes, Aldh1a1 and Fhl1. A previously reported in vitro TLX1 interacting protein, eIF4E, is also efficiently precipitated by streptavidin affinity capture of in vivo TLX1bio protein complexes. Thus, we believe that our adaptation of the FACS-based in vivo biotinylation tagging system provides a powerful tool for the characterization of transcription factor and other protein–protein complexes in mammalian cells.

Materials of Biotinylation Proteomics Protocol

Biotinylation Tagging Vectors

Starting material plasmids: the SIVmac1A11-based pCL20cSLFR MSCV-GFP lentiviral vector was provided by Arthur Nienhuis; coding sequences for a 23-amino acid biotinylation peptide tag (see Note 1) and the GFP-BirA fusion gene were obtained from the pTRIP/BPcter/IRES/EGFPBirA plasmid.
SIV lentiviral vectors: pCL20cSLFR MSCV-TLX1^bio-IRES-YFP expressing the TLX1 homeodomain transcription factor containing a COOH-terminal biotinylation peptide tag linked to a downstream YFP reporter on a bicistronic transcript; pCL20cSLFR MSCV-GFP-BirA expressing a fusion protein consisting of a GFP reporter and the bacterial BirA biotin ligase (GFP-BirA). The pCL20cSLFR MSCV-IRES-YFP and pCL20cSLFR MSCV-GFP-BirA plasmids can be obtained from the authors upon request.
Restriction and modifying enzymes for PCR amplification and subcloning of genes of interest.
Plasmid DNA purification kits: QIAquick Gel Extraction Kit, QIAGEN Plasmid Maxi Kit.

Production and Titration of Vector Particles

Transient Transfection

293T/17 (293T) human embryonic kidney cell line.
293T cell culture medium: Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4.5 g/L glucose, 2 mMl-glutamine, 50 IU/mL penicillin, 50 mg/mL streptomycin, 10% heat-inactivated fetal bovine serum (FBS). Store at 4°C and warm up to 37°C before use.
Phosphate-buffered saline without Ca²⁺ and Mg²⁺(PBS): 137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄ (adjust pH to 7.4 if necessary, using 1 N HCl or 1 N NaOH).
Solution of trypsin and ethylenediamine tetraacetic acid (trypsin-EDTA): 0.05% trypsin, 0.53 mM EDTA in PBS.
pCL20cSLFR MSCV-IRES-YFP biotinylation tagging SIV lentiviral vector plasmid DNA containing the gene of interest; pCL20cSLFR MSCV-GFP-BirA SIV lentiviral vector plasmid DNA.
SIV lentiviral packaging plasmid DNAs.
Vesicular stomatitis virus G (VSV-G) glycoprotein envelope plasmid DNA.
2.5 M CaCl₂: Dissolve 183.7 g CaCl₂ dihydrate (tissue culture grade) in deionized water. Bring the volume up to 500 mL and filter–sterilize using a 0.22-mm nitrocellulose filter. Store at −20°C.
2× N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES)-buffered saline (2× HBS): 50 mM HEPES, 280 mM NaCl, 1.5 mM Na₂HPO₄. Titrate to pH 7.05 with 5 N NaOH. Filter-sterilize using a 0.22-mm nitrocellulose filter. Store as single use aliquots at −20°C.

Collection and Concentration of Vector Particles

0.45-μm pore-size filter units: Stericup 150-mL unit, low protein binding Durapore (PVDF) unit.
Ultracentrifuge, polycarbonate 70-mL ultracentrifuge bottles and caps.

Titration of Vector Particles

HT1080 human fibrosarcoma cells.
HT1080 cell culture medium: same as 293T cell culture medium.
Polybrene stock solution (1,000×): 8 mg/mL in sterile, deionized water. Aliquot and store at −20°C.
Analytical flow cytometer.

Generation of Cells Stably Expressing Biotin-Tagged Proteins

Replication-defective SIV lentiviral vector particles.
Target cells.
Suspension cell culture medium: Iscove's modified Dulbecco's medium (IMDM) supplemented with 2 mM l-glutamine, 50 IU/mL penicillin, 50 mg/mL streptomycin, 10% heat-inactivated FBS.
Polybrene stock solution.
Fluorescence-activated cell sorter (see Note 3).

Cell Fractionation

Phenylmethylsulphonyl fluoride (PMSF): 0.5 M in dimethyl sulfoxide. Store at −20°C.
Dithiothreitol (DTT): 0.5 M in deionized water. Store at −20°C.
Protease inhibitors cocktail tablets.
PhosSTOP phosphatase inhibitor cocktail tablets (Roche Diagnostics Corp.).
Hypotonic wash buffer: 10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM PMSF, 0.5 mM DTT.
NP40 nuclear extraction (NP40 NE) buffer: 20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 20 mM KCl, 0.25 M NaCl, 0.1% NP40, 5 mM EDTA, 1 mM PMSF, 0.5 mM DTT, protease inhibitors, phosphatase inhibitors.
Other solutions: PBS; 0.4% trypan blue in normal saline.
(Optional) Sonifier.

Streptavidin Affinity Precipitation and Mass Spectrometry Analysis

Streptavidin sepharose beads.
Chicken albumin: 20 mg/mL in deionized water. Store at −20°C.
HENG buffer: 20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 0.25 mM EDTA.
NP40 T buffer: 70 mM Tris–HCl, pH 8.0, 25% glycerol, 1.5 mM MgCl₂, 20 mM KCl, 0.25 M NaCl, 0.1% NP40, 5 mM EDTA, 1 mM PMSF, 0.5 mM DTT.
Tris–glycine SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels, and associated running and loading buffers.
SDS-PAGE staining reagents.
In-gel tryptic digestion reagents.
ZipTip.
Elution buffer: 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) in Milli-Q^® grade water.
a-cyano-4-hydroxycinnamic acid: 10 mg/mL in 50% ACN, 0.05% TFA.
Mass spectrometer.

Methods of Biotinylation Proteomics Protocol

Biotinylation Tagging Vectors

Subclone the gene of interest linked to a biotinylation peptide tag upstream of the IRES-YFP cassette in the pCL20cSLFR MSCV-IRES-YFP SIV lentiviral vector (see Notes 1 and 4).
Extract plasmid DNA using plasmid DNA purification kits.

Production and Titration of Vector Particles

293T (293 human embryonic kidney cells expressing simian virus 40 [SV40] large tumor [T] antigen) cells are highly transfectable such that transient cotransfection with the self-inactivating SIV lentiviral vectors, packaging plasmids and envelope plasmids yields high-titer, replication-defective vector particles. Lentiviral vectors pseudotyped with the VSV-G glycoprotein have a broad host-cell range and can be utilized to transduce all cell types.

Transient Transfection

Propagate 293T cells in 293T cell culture medium at 37°C in a humidified atmosphere with 5% CO₂.
Passage the cells every 3–4 days by splitting them one fourth to one eighth. Remove the medium and rinse the cells with PBS. Remove the PBS and add enough trypsin-EDTA to cover the cells. Incubate the plate at room temperature until the cells round up and detach. Add an equal volume of cell culture medium to inactivate the trypsin. Collect the cells and centrifuge for 5 min at 375×g. Resuspend the cells in fresh cell culture medium for plating.
Transfect 293T cells with plasmid DNA using the calcium phosphate precipitation method. On the day before transfection, plate 293T cells in 7 mL of cell culture medium at a density of 5×10⁶ cells per 100-mm tissue culture dish.
Prepare a mixture of 20 mg of plasmid DNA composed as follows: 10 mg of pCL20cSLFR MSCV-IRES-YFP biotinylation tagging SIV lentiviral vector containing the gene of interest (or pCL20cSLFR MSCV-GFP-BirA SIV lentiviral vector), 6 mg of pCAG-SIVgprre plus 2 mg of pCAG4-RTR-SIV packaging plasmids, and 2 mg of the pMD.G VSV-G glycoprotein envelope plasmid (see Note 5). Bring the volume up to 400 mL with sterile, deionized water. Add 100 mL of 2.5 M CaCl₂ and mix. Add the DNA/CaCl₂ solution dropwise to 500 mL of 2× HBS in a 15-mL conical tube. Use a second pipettor and a 2-mL pipet to bubble the 2× HBS as the DNA/CaCl₂ solution is added. Vortex immediately for 5 s and incubate at room temperature for 20 min. Add the 1 mL of DNA/calcium phosphate mixture directly to each 100-mm dish while swirling (see Note 6). Incubate the cells at 37°C overnight (16 h). Change the medium and culture for 24–48 h (this method usually results in transfection of 50–80% of the cells).

Collection and Concentration of Vector Particles

Collect the culture medium containing vector particles 24–48 h after medium change. Centrifuge at 2,000×g for 10 min to remove cellular debris and filter through a 0.45 mm pore-size filter (depending on the volume, use a 150-mL filter unit or a small filter unit attached to a 5-mL syringe). Use directly for transductions or aliquot and store at −80°C (see Note 7). Several procedures have been developed to concentrate lentiviral vector particles. The choice of concentration protocol depends on the envelope selected to pseudotype the particle and the quantities of particles to be produced. The stability of the VSV-G envelope protein allows the generation of high-titer lentiviral vector particles by ultracentrifugation, as described here.

Ultracentrifuge the vector particles at 50,000×g and 4°C for 90 min.
Discard supernatant. Using gentle pipetting, resuspend the pellet in ~100 mL of medium appropriate for the downstream application. To facilitate complete resuspension, vortex gently overnight at 4°C (see Note 8).
To remove cellular debris, centrifuge concentrated vector particles at 10,000×g and 4°C for 5 min in a microcentrifuge. Collect the supernatant containing the vector particles, aliquot and freeze at −80°C (see Note 9).

Titration of Vector Particles

The HT1080 human fibrosarcoma cell line can be used to determine the titer of the lentiviral vector particles.

Propagate HT1080 cells in HT1080 cell culture medium at 37°C in a humidified atmosphere with 5% CO₂.
4–6 h prior to titrating the vector particles, plate 2.5×10⁵ HT1080 cells into each well of a 6-well tissue culture dish.
Prepare serial dilutions of each vector preparation (e.g., 100, 10⁻¹, and 10⁻² for unconcentrated vector particles; 10⁻², 10⁻³, and 10⁻⁴ for concentrated vector particles) using cell culture medium, in a final volume of 1 mL per dilution. Add 1 mL of polybrene stock solution to each dilution. Remove the medium from the cells and add 1 mL of each dilution to each well. Incubate at 37°C for 4 h.
Remove the vector particles after the 4-h transduction and replace with 2 mL of fresh cell culture medium. Return to the CO₂ incubator and incubate at 37°C.
After 48 h, determine the relative end-point vector titer (in transducing units per mL [TU/mL]) by flow cytometric analysis. Evaluate GFP or YFP expression on an analytical flow cytometer equipped with 488 nm excitation wavelength and a 530/30-nm bandpass (BP) filter.
To determine vector titer, use the following equation: Vector titer = number of HT1080 cells × % fluorescent protein-positive cells×dilution factor (see Note 10).

Generation of Cells Stably Expressing Biotin Tagged Proteins

The following protocol is used to transduce suspension cell lines with the recombinant biotinylation tagging lentiviral vectors. For adherent cells, the protocol for titration of vector particles is used.

Propagate cells (e.g., SupT1) in suspension cell culture medium at 37°C in a humidified atmosphere with 5% CO₂.
Prepare an appropriate dilution of vector particles in 1 mL of cell culture medium containing 1 mL of polybrene stock solution. Resuspend 1–2×105 cells in the diluted vector particles in a 14-mL conical polystyrene centrifuge tube.
Centrifuge the mixture of cells and vector particles at 600×g and 15°C for 2 h. Resuspend the cell pellet in 0.5 mL of fresh cell culture medium and incubate overnight. Repeat the above centrifugation-enhanced transduction once a day for 2 days.
Culture for 10 days. Isolate target cells stably expressing the appropriate fluorescent protein(s) on a fluorescence-activated cell sorter equipped with 488 nm excitation wavelength (see Note 11). Evaluate GFP and YFP fluorescence with a 510/20-nm BP filter and a 550/30-nm BP filter, respectively, separated by a 525-nm shortpass dichroic mirror. To determine correct settings for fluorescence compensation, use cells expressing GFP or YFP alone as single color controls.

Cell Fractionation

The protocol described below is a variation of the Dignam procedure for the preparation of nuclear extracts for human tissue culture cells that has been modified for T-ALL cell lines (e.g., SupT1). The standard procedure is based on a starting cell number of 2×10⁸ cells and can be adjusted to accommodate 2×10⁹ cells by increasing the buffer volumes at steps 2 and 3 by a factor of 4. All solutions are kept on ice; prior to cell harvesting, freshly thawed PMSF and DTT, and freshly prepared protease and phosphatase inhibitors are added to the prechilled buffers. Centrifugations are performed at 4°C.

Harvest the cells by centrifuging at 600×g for 10 min.
Wash once in 10 mL of PBS and centrifuge at 600×g for 10 min.
Wash once in 10 mL of hypotonic wash buffer and centrifuge at 600×g for 5 min.
Remove supernatant immediately and discard; resuspend the pellet in 3 mL of hypotonic wash buffer.
Subject the cells to hypotonic shock by incubating for 30 min.
Check the efficiency of cell lysis under a microscope by staining an aliquot with trypan blue.
Centrifuge homogenate at 3,300×g for 15 min.
Remove supernatant (mostly composed of cytosolic and loosely-associated nuclear proteins) and adjust salt concentration by adding an equal volume of NP40 NE buffer. Freeze sample at −80°C or keep on ice until streptavidin affinity precipitation.
Resuspend the pellet in 3 mL of NP40 NE buffer.
Incubate for 30 min on ice to extract nuclear proteins; during the incubation period, shear DNA either by mild sonication or by passing the material through a 25-gauge needle.
Freeze sample at −80°C or keep on ice until streptavidin affinity precipitation.

Streptavidin Affinity Precipitation and Mass Spectrometry Analysis

Streptavidin affinity precipitation is performed essentially as described (see Note 12).

Wash 120 mL of streptavidin sepharose beads (for two samples) with 1 mL of HENG buffer. Centrifuge at 10,000×g for 20 s. Resuspend the beads in 500 mL of HENG buffer containing 200 mg/mL of chicken albumin and incubate on a rotating platform for 1 h at room temperature. Centrifuge and resuspend the beads in 65 mL of HENG buffer.
Centrifuge the samples at 25,000×g for 30 min. To decrease nonspecific binding, transfer supernatants to new tubes even if there is no visible insoluble material.
Add 30 mL of streptavidin bead suspension per sample and incubate on a rotating platform for 1 h at 4°C.
Wash the sample twice by resuspending in 1 mL of NP40 T buffer and centrifuging at 10,000×g for 20 s.
Wash the sample three times by resuspending in 1 mL of NP40 T buffer, incubating on a rotating platform at room temperature for 5 min, and centrifuging at 10,000×g for 20 s.
Resuspend the material in 2× loading buffer and fractionate by SDS-PAGE.
Stain gel with colloidal blue. Excise discretely stained bands that are evident only in the presence of biotinylated protein of interest.
Subject each band to in-gel tryptic digestion. Desalt and concentrate resultant peptides through a ZipTip, and elute in 5 mL of elution buffer.
Load 1 mL of peptides and 1 mL of a-cyano-CHCA matrix onto a target plate.
Conduct peptide mass fingerprinting using MALDI-TOF mass spectrometry. Perform protein database searches using Mascot software.
Confirm the identity of interacting proteins revealed by mass spectrometry using Western blot analysis.

Notes of Biotinylation Proteomics Protocol

More recently, we have constructed an NH₂ -terminal biotin-tagged TLX1 (bioTLX1) modeled after the ^bioNanog homeo-domain protein and other biotin-tagged transcription factors described by Orkin and colleagues, which is based on the 15 amino acid AviTag™ sequence (GLNDIFEAQKIEWHE) that is very efficiently biotinylated in vivo by BirA.
The 293T/17 cell line is a clone of the 293T (293tsA- 1609neo) human embryonic kidney cell line, which was selected for high transfectability and capability of producing high-titer vector stocks. The 293T cell line is a derivative of 293 cells into which the simian virus 40 (SV40) large T antigen gene was inserted. 293 cells express the adenovirus serotype 5 E1A 12S and 13S gene products, which strongly transactivate transcription from expression vectors containing the human CMV enhancer-promoter. Expression of the SV40 large T antigen by 293T cells may stimulate extrachromosomal replication of plasmids containing the SV40 origin of replication during transient transfection.
Standard detection filters supplied with most commercial flow cytometers are unable to distinguish between GFP and YFP signals.
Common recombinant DNA techniques are used for plasmid DNA preparations, restriction enzyme digestions, PCR amplifications, and subclonings. Detailed protocols for each technique can be obtained from commercial sources, as well as from various standard molecular biology manuals. Accordingly, these techniques have not been described here.
The pCL20cSLFR MSCV-GFP SIV-based vector system is modeled after “third generation” lentiviral vector systems that have incorporated features to enhance safety and improve the efficiency of vector particle production. Specifically, several viral accessory proteins have been eliminated from the pCAG-SIVgprre gag-pol packaging construct, and the LTRs have been modified to contain the human CMV enhancer– promoter and to eliminate Tat dependence during vector production (5' LTR) and to be self-inactivating (3' LTR) upon integration. Because both the expression of the pCL20cSLFR MSCV-GFP SIV lentiviral vector backbone as well as the gag and pol genes encoded by the pCAG-SIVgprre packaging construct are dependent on transcomplementation by the Rev protein, use of this system requires cotransfection with a rev expression construct (e.g., pCAG4-RTR-SIV).
Vector titer depends on the vector backbone design, the size and nature of inserted sequences as well as the efficiency of transfection. To achieve optimal transfection efficiency, use an exponentially growing culture (50–70% confluent) of 293T cells for transfection and make sure that the cells form a uniform monolayer upon plating. Transfection efficiency is also affected by the quality of the plasmid DNA used. Commercially available plasmid DNA purification kits usually yield highly purified endotoxin-free supercoiled plasmid DNA (traditionally obtained by purifying on two separate cesium chloride gradients). Sterilize the plasmid DNA by ethanol precipitation, resuspend the air-dried pellet in sterile, deionized water, and determine its concentration and quality by spectrophotometric analysis and gel electrophoresis. Pay special attention to the pH of the 2× HBS solution, which should be between 7.05 and 7.12. Upon addition of the transfection mixture to the cells, a fine precipitate should develop within a few minutes.
pH and temperature fluctuations as well as freeze-and-thaw frequency could have an impact on the stability of vector particles, and hence, vector titer. Minimize pH changes by adding HEPES at a final concentration of 10 mM to buffer the cell culture medium. Once collected, vector particles should be kept on ice at all times. For future use, they should be stored in aliquots at −80°C. Avoid repeated freezing and thawing.
Small (~2 to 5-mm-diameter) pellets should be visible after concentration by centrifugation. Expect a 50–75% recovery following vector concentration.
The vector particle preparation can be further purified if necessary by centrifuging through a sucrose cushion.
When reporter proteins are evaluated by flow cytometry, it is advantageous to wait at least 5 days before analyzing the transduced cells. This minimizes the contribution of false positive signals due to pseudotransduction, which is the direct transfer of reporter protein (adhered to vector particles or incorporated into vector particles) to the target cells; this is particularly problematic for VSV-G glycoprotein-pseudotyped vectors. Note that it has also been shown that transgenes can be efficiently transiently expressed from unintegrated lentiviral vectors during this timeframe (10–14 days).
When the target cells already express the protein of interest, sort YFP/GFP-positive cells that express a subendogenous level of the biotin-tagged protein so as to avoid perturbing any existing protein–protein network interactions. To further minimize competition between endogenous and biotin-tagged proteins under these circumstances, we have recently adapted the shRNA-expressing pLKO.1-puro lentiviral vector, by substituting the puromycin N-acetyltransferase gene with the cyan fluorescent protein (CFP) gene so that simultaneous knockdown of endogenous transcripts can be achieved by sorting CFP-positive cells.
While single-step purification to generate protein complexes sufficiently clean to be analyzed by mass spectrometry is a strength of the in vivo biotinylation approach, it might be advantageous to perform tandem-affinity purifications in some experiments. Elegant studies carried out by Orkin and colleagues in murine embryonic stem cells to elaborate the transcriptional interaction network involving nine transcription factors, including the Nanog homeoprotein, have validated and demonstrated the utility of in vivo biotinylation of tagged proteins and streptavidin affinity capture to identify downstream targets on a global scale by ChIP-on-chip. In those studies, both single-step capture on streptavidin beads as well as tandem purification with anti-Flag immunoprecipitation followed by capture with streptavidin were carried out. For example, candidate Nanog-interacting proteins fell into three groups: the first group included proteins present in at least two of three independent one-step purifications and tandem purification; the second group included proteins that may be part of unstable or transient complexes that dissociate during tandem purification; and the third group contained proteins that were "masked" in one step purifications but were recovered in the tandem procedure. For this reason, we have also included NH₂-terminal Flag tags on our recombinant proteins, which can be used for this purpose.