We thank Weixan Lu for technical assistance in the culture and maintenance of the Sf9 cell stocks. L.A.Y. was supported by a Medical Research Council (MRC) graduate studentship. R.J.C.G was a Royal Society University Research Fellow. The Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust Core Award Grant Number 090532/Z/09/Z.

This protocol assumes that the mouse kindlin-3 gene (FERMT3) has been successfully cloned into a vector downstream of the very late p10 promoter and that the vector possesses flanking baculovirus sequences to permit recombination with the bacmid BAC10KO:1629 developed by I.M. Jones and colleagues28. For this protocol, the kindlin-3 gene was cloned into pOPINE29 and the primers and cloning strategy used can be found described elsewhere12. The plasmid is engineered so that the FERMT3 gene (kindlin-3) is under the control of the p10 baculoviral promoter and the vector contains 5&#8242; UTR/ORF603 and ORF 1629 and encodes a C-terminal His6-tag for downstream purification29.
1. Insect Cell Culture and Maintenance
<ol>
	<li>Prior to recombinant baculovirus amplification, Spodoptera frugiperda cells adapted to suspension culture (Sf9 cells) should be grown and maintained. Insect cells should always be handled using aseptic techniques in a dedicated tissue culture fume hood.</li>
	<li>Suspension cultures of insect cells are incubated in Sf-900 II (SFM) serum free liquid medium supplemented with 100 &#956;g/ml penicillin and 100 &#956;g/ml streptomycin in flasks at 27 &#176;C with shaking at 100 rpm.</li>
	<li>Maintain the insect cell culture density range between 1 x 106&#160;- 1 x 107&#160;cells/ml by splitting and diluting the cell culture with fresh Sf-900 II media. 
	Note: healthy cells should look uniform in size and should be spherical in shape.</li>
	<li>Count the cells in a sample volume using a hemocytometer and light microscopy to calculate the culture cell density.</li>
</ol>
2. Generation of Recombinant Baculovirus
<ol>
	<li>Culture and maintain Sf9 cells in suspension using Sf-900 II media supplemented with 100 &#956;g/ml penicillin and 100 &#956;g/ml streptomycin. For baculovirus generation, insect cells should be cultured to a density range of 5 x 105&#160;-&#160;1 x 106 cells/ml.</li>
	<li>Seed approximately 1 x 106 Sf9 cells per well of a sterile 6-well tissue culture plate in 2 ml of Sf-900 II media supplemented with 100 &#956;g/ml penicillin and 100 &#956;g/ml streptomycin. Leave the Sf9 cells at RT in the fume hood to adhere to the base of the plastic wells and thus form a monolayer.</li>
	<li>Perform recombinant baculovirus generation by cotransfecting bacmid DNA and plasmid DNA onto monolayer culture.
	<ol>
		<li>For each transfection, mix 1-2 &#956;g of purified pOPINE-mFERMT3, which possesses the ORF1629 baculovirus elements, with 0.5 &#956;g of purified BAC10KO:1629 in 100 &#956;l of Sf-900 II SFM without antibiotics (Solution A).</li>
		<li>In a separate tube, dilute 6 &#956;l of Cellfectin II Reagent with 100 &#956;l of Sf-900 II SFM without antibiotics for each transfection reaction (Solution B). A &#8216;master mix&#39; can be created here, for reduced liquid handling, if many transfections are required.</li>
		<li>Mix the two solutions (A and B, approximately 200 &#956;l) and incubate at RT for 20 min&#160;to form a lipid-DNA complex.</li>
		<li>Dilute the lipid-DNA complexes with 800 &#956;l Sf-900 II SFM without antibiotics. Carefully aspirate the Sf9 cell monolayer media and carefully pipette the solution A/B and media on top of the Sf9 monolayer.</li>
		<li>Incubate the transfected cells in a humidified incubator at 27 &#176;C O/N and add a further 1 ml of Sf-900 II SFM without antibiotics to each monolayer culture the following day. Incubate cells at 27 &#176;C for a further 5 days.</li>
	</ol>
	</li>
	<li>Harvest the recombinant baculovirus directly from the culture medium (approximately 2 ml in total) and transfer to a clean centrifuge tube (for example a 15&#160;ml Falcon tube).
	<ol>
		<li>Clarify any Sf9 cell debris by centrifugation at 1,000 x g for 5 min&#160;at RT. Transfer the virus, which is in the resulting supernatant and denoted P1, to a clean tube and store at 4 &#176;C in the dark until use. At this stage the remaining Sf9 monolayer can be used to assess recombinant virus generation by assessing the presence of recombinant kindlin-3 in the insect cells.</li>
	</ol>
	</li>
	<li>Resuspend the monolayer with 0.5 ml PBS and dilute a 10&#160;&#956;l sample with equal volumes of 2x&#160;SDS-PAGE loading buffer. Heat the samples at &#62;95 &#176;C for at least 10 min.
	<ol>
		<li>Sonicate the sample for 1 sec&#160;at 10% amplitude using a micro-sonicator tip if it is too viscous for proper gel loading.</li>
	</ol>
	</li>
</ol>
3. Amplification of Recombinant Baculovirus
<ol>
	<li>For the amplification of virus in suspension, use a cell density of 1.4 x 106 cells/ml and this should occupy 1/20&#160;of the total flask volume (i.e. 50 ml culture in a 2&#160;L&#160;flask).</li>
	<li>Achieve the amplification of the recombinant virus by infecting the insect cell culture with the P1 viral stock at a multiplicity of infection (MOI) of 0.1 using the following formula; 
	<img alt="" fo:content-width="4in" src="/files/ftp_upload/51206/51206eq1.jpg" width="400" /> 
	Note: P1 virus generation can be assumed to have an expected viral titer&#160;of 1 x 107 pfu/ml. However, a plaque assay can be performed prior to this stage.</li>
	<li>Incubate the P1-infected insect culture at 27 &#176;C with shaking at 100 rpm&#160;for 3 days (72 hr).</li>
	<li>Harvest the virus by separating the cells from the media by centrifugation at 1,000 x&#160;g for 5 min&#160;at RT. Save the resulting cell pellet at this stage for confirmation of recombinant virus production by assessing kindlin-3 protein expression by SDS-PAGE and western blotting.</li>
	<li>Transfer the clarified virus-enriched media to a clean tube and store at 4 &#176;C in the dark until use. This viral stock is denoted as P2. 
	Note: A viral titer of 2 x 108 pfu/ml (or an amplification rate of 100 pfu/cell) can be anticipated.</li>
</ol>
4. Expression of kindlin-3 in Baculovirus-Infected Sf9
<ol>
	<li>Grow an adequate volume of Sf9 cells cultures in suspension in Sf-900 II SFM supplemented with 100 &#956;g/ml penicillin and 100 &#956;g/ml streptomycin, prior to large-scale recombinant kindlin-3 expression for purification. Incubate the suspension cultures at 27 &#176;C with shaking at 100 rpm&#160;and with a total culture volume:flask volume ratio of 1:5.</li>
	<li>Infect suspension cultures of Sf9 at a density of 2 x 106 cells/ml.</li>
	<li>Supplement Sf9 cultures with a final concentration of 1% (v/v) Fetal Bovine Serum (FBS) followed with amplified recombinant virus (P2-viral stock) to give a MOI of 1. Incubate the infected cultures at 27 &#176;C with shaking at 100 rpm.</li>
	<li>Harvest recombinant kindlin-3-CHIS6-expressing Sf9 cells 72 hr&#160;post-infection by centrifugation at 1,000 x g&#160;and the resulting cell pellet stored at -20 &#176;C until use, or -80 &#176;C for long term storage.</li>
</ol>
5. Purification of Recombinant Kindlin-3
<ol>
	<li>Thaw frozen baculovirus-infected insect cell (Sf9) pellets expressing recombinant kindlin-3 on ice.</li>
	<li>Resuspend the thawed cell pellet with lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1% (v/v) Tween-20), supplemented with EDTA-free protease inhibitor cocktail and 1,000-2,000 U of DNase1. 
	Note: Alternatively, modified phosphate buffered saline (PBS) can also be used, where the NaCl concentration is adjusted to 500 mM to prevent nonspecific interactions between endogenous Sf9 cell proteins and the immobilized metal affinity column used for downstream purification (see below).</li>
	<li>Lyse the cells by incubating the resuspended cells with the detergent and vortexing. Sonicate (40% amplitude, 10 cycles of 10 sec pulse followed by 10 sec cooling) the sample for further cell disruption in an ice bath or alternatively use a Dounce homogenizer.</li>
	<li>Clarify the lysate by centrifugation at 48,000 x g for 1 hr&#160;at 4 &#176;C. Load the resulting supernatant onto a HisTrap column (5 ml column volume), pre-equilibrated with lysis buffer, at 4 &#176;C at a rate of 1 ml/min. 
	Note: Alternatively, the clarified lysate can be incubated with a 1-5 ml bed volume of pre-equilibrated nickel sepharose beads (e.g. Ni Sepharose 6 fast flow) at 4 &#176;C for 1-2 hr. A column of Ni sepharose can be formed after the binding step by using a gravity flow column.</li>
	<li>Wash the column with 10 column volumes of wash buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole) to remove unbound proteins.</li>
	<li>Use a linear imidazole gradient from 10-500 mM at a rate of 10 mM/ml (or in 10 column volumes) using an &#196;kta FPLC to elute the bound recombinant kindlin-3-CHIS6.</li>
	<li>Fractionate the elution into 0.5-1 ml fractions using the &#196;kta FPLC, with those fractions containing kindlin-3-CHIS6 usually eluting at an imidazole concentration of 300 mM.</li>
	<li>Assess the protein composition of the eluant by SDS-PAGE and for first-time purifications confirm by western blotting using an anti-His6 antibody or anti-mouse kindlin-3 antibody.</li>
	<li>Pool the fractions containing kindlin-3 and buffer exchange into 20 mM Tris-HCl, pH 7.5, 200 mM NaCl via a series of dilutions into buffer and sample concentrations using a centrifugal protein concentrator with a 50 kDa molecular weight cut-off (MWCO) at 4 &#176;C. 
	Note: Alternatively, dialyze the protein solution using Slide-A-lyzer Dialysis Cassette with a MWCO of 30 kDa at 4 &#176;C for 4 hr&#160;to O/N&#160;into 20 mM Tris-HCl, pH 7.5, 200 mM NaCl. 
	Note: for ion exchange (see below) a lower concentration of NaCl, i.e. 50 mM, can be and has been used for this step.</li>
	<li>Apply the buffer-exchanged protein solution onto a pre-equilibrated HiTrap heparin HP column (5 ml column volume) using an &#196;kta FPLC at a rate of 0.5 ml/min. 
	Note: The bound kindlin-3 is eluted using a linear NaCl gradient (0.2 M NaCl to 1 M NaCl) in the same buffer, increasing at a rate of 10 mM/ml. Kindlin-3-CHIS6 is expected to elute at ~0.6 M NaCl.</li>
	<li>Fractionate the elution into 0.5-1 ml fractions and assess the protein composition by SDS-PAGE and western blotting, if appropriate. 
	Note: one can expect a protein purity of close to 95%, as assessed by SDS-PAGE.</li>
	<li>Pool fractions containing kindlin-3 and concentrate using a centrifuge protein concentrator with a 50 kDa MWCO to a final volume of 0.5-2 ml.</li>
	<li>In a final step, polish the concentrated protein and buffer exchange the protein using size exclusion chromatography (SEC).
	<ol>
		<li>Apply the purified protein onto a Superdex S200 (16/60) or (10/30) pre-equilibrated in 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM DTT at a rate of 1 ml/min or 0.5 ml/min depending on column size used. 
		Note: Size exclusion chromatography can also be performed in phosphate-buffered saline (PBS) if required.</li>
		<li>Purify the proteins according to size by applying buffer onto the column at a rate of 1 ml/min or 0.5 ml/min, depending on column size used. 
		Note: The protein eluting from the column is fractionated and monitored using the absorbance at 280 nm.</li>
		<li>A single absorbance peak should be expected from SEC, which is fractionated and assessed by SDS-PAGE to determine homogeneity, and is typically &#62;95% pure after this step.</li>
	</ol>
	</li>
	<li>Concentrate the purified kindlin-3-CHIS6 using a centrifuge protein concentrator with a 50 kDa MWCO to ~15 mg/ml, as assessed spectrophotometrically using a calculated extinction coefficient (&#949;) of 109,320 M-1 cm-1 (assuming all cysteine residues are reduced).</li>
	<li>For storage at -20 &#176;C and long term storage at -80 &#176;C, aliquot the protein into PCR tubes and flash-freeze the samples in liquid nitrogen. Alternatively, the protein can be used directly for investigation using a number of biochemical and biophysical techniques.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+production+of+proteins+in+insect+and+mammalian+cells+for+structural+biology.">Recent advances in the production of proteins in insect and mammalian cells for structural biology.</a> J Struct. Biol. 172, 55-65 .</li><li>Wasilko, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+titerless+infected-cells+preservation+and+scale-up+(TIPS)+method+for+large-scale+production+of+NO-sensitive+human+soluble+guanylate+cyclase+(sGC)+from+insect+cells+infected+with+recombinant+baculovirus.">The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus.</a> Protein Expr. Purif. 65, 122-132 (2009).</li><li>Yates, L. A., Fleurd&#233;pine, S., Rissland, O. S., De Colibus, L., Harlos, K., Norbury, C. J., Gilbert, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+for+the+activity+of+a+cytoplasmic+RNA+terminal+uridylyl+transferase.">Structural Basis for the activity of a cytoplasmic RNA terminal uridylyl transferase.</a> Nat. Struct. Mol. Biol. 19, 782-787 (2012).</li><li>Sainsbury, S., Ren, J., Saunders, N. J., Stuart, D. I., Owens, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+analysis+of+CrgA,+a+LysR-type+transcriptional+regulator+from+pathogenic+Neisseria+meningitidis+MC58.">Crystallization and preliminary X-ray analysis of CrgA, a LysR-type transcriptional regulator from pathogenic Neisseria meningitidis MC58.</a> Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 64, 797-801 (2008).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell+adhesion+and+migration+by+Kindlin-3+cleavage+by+calpain.">Regulation of cell adhesion and migration by Kindlin-3 cleavage by calpain.</a> J. Biol. Chem. 287, 40012-40020 .</li></ol>

The authors have nothing to disclose.

Baculovirus expression systems are becoming increasingly popular and an important tool for the production of milligram quantities of recombinant protein for protein characterization using biophysical studies, including X-ray crystallography. Despite being more experimentally demanding the baculovirus expression systems offer several advantages over E. coli one of which is a near-native environment for proteins of eukaryotic origin e.g. the presence of appropriate chaperones and opportunity for post-translational modification. In our own efforts to express kindlin-3, alternative expression hosts were used including mammalian cell-lines and bacterial expression strains (unpublished observations). Generally, the many E. coli strains tested produced very small quantities of recombinant kindlin-3 (~0.5 mg/L of culture; unpublished observations). However, the baculovirus-driven expression in insect cells was particularly effective and, in comparison to transient expression in mammalian cells, more amenable to generating the large biomass required for isolating a milligrams of recombinant cytoplasmic proteins (unpublished observations). We speculate that the presence eukaryotic chaperones may allow the efficient production of kindlin-3.
The baculoviridae infect insect cells and for recombinant protein expression the baculovirus used in this work is based upon the Autographa californica nuclear polyhedrosis virus (AcNPV). In nature the AcNPV, which infects the Autographa californica (alfalfa lopper) insect larvae, requires the polyhedrin protein to form occlusions whereby the virons are encapsulated in a crystalline protein matrix thus providing the necessary protection for their release. In cultured cells the formation of occlusion bodies is not required for replication and is therefore dispensable. In the case of expressing foreign proteins the polyhedrin protein gene can be replaced in a recombinant AcNPV with the gene for the protein of interest. AcNPV can infect other Lepidopteron species and for the purposes of recombinant protein expression army worm Spodoptera frugiperda pupal ovary cells are used. In the approach outlined here, the AcNPV bacmid (BAC10) is engineered so that an essential viral gene, ORF1629, is inactivated by the insertion of chloramphenicol acetyl transferase resulting in a knock-out bacmid (BAC10:KO1629), such that it is unable to form infectious baculovirions28. Cotransfection of Sf9 cells with linearized BAC10:KO1629 and the FERMT3-containing transfer vector repairs the inactive ORF1629, through transposition, resulting in a viable genome that has also incorporated the FERMT3 gene under the control of the polyhedrin promoter28.
We describe a purification protocol for the isolation of highly pure recombinant mouse kindlin-3 via a three step chromatographic approach. The methods used here could easily be applied to other His-tagged proteins. We employed an ion exchange step to further purify kindlin-3 but we believe that this is a further pseudo-affinity step as kindlin-3 possesses a large number of basic residues, including a poly-lysine stretch within its F1 domain. Additionally, kindlin-3 is considered to bind to and interact with the cytoplasmic face of the plasma membrane, where it functions, and therefore we predict that the clustering of basic residues will allow the protein to counter the negatively charged membrane.
The buffers described in the purification protocols are considered standard and are frequently used in structural biology. The thermofluor assay (Figure&#160;5) demonstrates that kindlin-3 is stable in&#160;most buffer conditions above pH 6.0. This was particularly useful and important for informing our experiments when studying kinldin-3:&#946;1A tail interaction by NMR, which yielded excellent spectra at pH 6.1 with low concentrations of NaCl12.
Before any biophysical study can be undertaken, it is important to demonstrate that the purified protein of interest is indeed correctly folded and is functionally active. In a previous publication we demonstrated that the recombinant kindlin-3 expressed and purified using this method was a monomer and monodispersed in solution, as assessed by size-exclusion chromatography, dynamic light scattering, analytical ultracentrifugation and small angle X-ray scattering, and was also capable of binding and recognizing the membrane-distal NPxY and upstream Serine/Threonine cluster of &#946;1A cytoplasmic tails14, thus confirming that it behaves like the native protein, which is in line with previous cellular and physiological studies14,20,22. The use of a thermal stability assay is an additional way of suggesting the proper folding of a protein of interest,&#160;as incorrectly folded protein will result in a high fluorescence background due to exposed hydrophobic residues.
The kindlin family of proteins has been the focus of much attention since their unexpected role as essential coactivators of integrins in vivo was discovered. This has triggered much effort to express them recombinantly and resolve their structures. To date limited success has been reported in expressing milligram quantities of full length recombinant protein but we have here described the use of a baculovirus system that permits large scale expression at levels where structural studies become feasible. By generating large quantities of recombinant kindlin-3 we anticipate that this will aid further studies of this protein. The baculovirus-driven method and purification workflow described here for recombinant murine kindlin-3 could also be used to express and purify the other kindlin isoforms, which are also difficult to express and also possess poly-lysine stretches, and may be further adapted for other cytoplasmic proteins, such as nucleic acid binding proteins, that fail to express in bacterial strains.

Proteins of the kindlin family are a crucial component of focal adhesion assembly, and therefore essential for complex life. Kindlins, of which there are 3 isoforms in mammals (kindlin-1, kindlin-2, and kindlin-3), are considered coactivators of the extracellular receptors integrins alongside talin1. Integrin-mediated cell adhesion connects the cell surface to the extracellular matrix (ECM) in higher eukaryotes. It is a critical and common process in a plethora of physiological phenomena, which include tissue integrity, embryogenesis, bone metabolism, hemostasis, and immunity. Integrin-mediated cell adhesion is activated through inside-out signal transduction via the binding of talin and kindlin to the integrin &#946;-subunit cytoplasmic tails (CTs) at their conserved NPxY motifs. The biomedical importance of kindlin proteins extends however as far as the nucleus, where kindlin-2 has been shown in several recent reports to be involved in transcriptional control2,3.
Kindlins are multidomain proteins of approximately 75 kDa, hallmarked by the possession of a bipartite C-terminal FERM (4.1 band, ezrin, radixin, moesin) domain, which is interrupted by a pleckstrin homology (PH) domain at the center&#160;of its F2 subdomain4,5. Studies of the kindlin-2 and kindlin-3 PH domains showed that it binds to the lipid second messengers phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(4,5)-bisphosphate6-8. However, studies of the kindlin-1 PH domain show that it binds to PtdIns(3,4,5)P3 with a much lower affinity, which can be explained in kindlin-1 by an isoform-specific salt bridge preventing lipids from binding9. In addition, there is an ~100 amino acid loop inserted into the F1 domain of the kindlins that is predicted to be unfolded but binds to phosphatidylserine in the inner leaflet of the plasma membrane10,11. The kindlin FERM domain is considered homologous to the talin FERM domain, although the talin FERM domain does not possess a pleckstrin homology domain. Both kindlins and talin interact with NPxY motifs on the integrin &#946;-tails via the F3 region of their FERM domain, but kindlin binds to the membrane distal motif, while talin targets the membrane proximal one12-16. Kindlins and talin both in addition possess an N-terminal F0 domain with a ubiquitin-like fold that is not found in other FERM proteins11,17. Studies on the F0 domain of kindlin-2 have shown that it independently binds to phosphatidylinositol-(4,5)-bisphosphate enriched membranes17.
The kindlins exhibit paralogue-specific tissue expression patterns and nonredundant physiological functions. Kindlin-1 is primarily expressed in the epidermis, but also to a lesser extent the colon, stomach, and kidneys; kindlin-2 is ubiquitously expressed but is concentrated in striated and smooth muscle and is the only kindlin expressed in embryonic development4; and kindlin-3 is expressed in the hematopoietic tissues with the highest concentration of kindlin-3 found in megakaryocytes18. However, more recent studies have suggested that functional protein is expressed in endothelial tissues as well19.
Kindlin-3 is of acute medical interest due to its important physiological role in the blood. It is critical for platelet aggregation and spreading during thrombus formation20, leukocyte rolling in response to infection and inflammation21,22 and osteoclast podocyte formation in bone resorption23. Furthermore, depletion of kindlin-3 in humans leads to leukocyte adhesion deficiency type-III - a disease characterized by life-threatening bleeding disorders and recurrent bacterial infections20,24,25. Kindlin-3 knock-out studies in mice revealed the crucial function of the protein in cell adhesion. KIND3-/- mice display distinct phenotypes, such as severe bleeding due to inactive platelet integrins, severe osteopetrosis, and impaired leukocyte adhesion20,22, resembling symptoms in humans lacking kindlin-3.
High-resolution structural data on the kindlins, to date, has been restricted to individual subdomains such as the pleckstrin homology (PH) domain of kindlin-19 and kindlin-226,27 and the F0 domain of kindlin-111 and kindlin-217. Most of the subdomains of each kindlin polypeptide have however resisted cloning and structural analysis (Yates and Gilbert, unpublished observations), and studies of the full-length proteins have been hindered by the difficulty of expressing and purifying sufficient quantities using E. coli (unpublished observations and Harburger&#160;et al.14). There is considerable medical interest in kindlin-3 and its function, alongside the other two family members, and recently we generated milligram quantities of it by recombinant expression in Spodoptera frugiperda cells driven by baculovirus infection12. We therefore here describe methods for the production of milligram quantities of recombinant mouse kindlin-3 in insect cell culture, suitable for extensive structural studies and biochemical analysis.
In this protocol we make use of an engineered knockout bacmid (BAC10KO:1629) that is, alone, unable to produce viable virions28. The viral DNA is thus rescued by recombination with a transfer vector that in this case also includes the kindlin-3 gene (FERMT3) and results in the FERMT3 gene replacing the virus very late gene, which is highly expressed but redundant, resulting in a recombinant virus that expresses mouse kindlin-3 as part of the virus life cycle28. We identified this method for the production of kindlin-3 after attempts to express and purify it in other expression hosts proved prohibitively difficult (unpublished observations) but also due to the versatility of the pOPIN vector suite, which we used for cloning and that can deployed in many expression hosts29.

<table><tbody><tr><td>Sf-900 II serum free media (SFM) 1x liquid</td><td>Life Technologies</td><td>10902-096</td><td>store at 4 &amp;deg;C and warm to RT before use</td></tr><tr><td>Cellfectin II Reagent</td><td>Invitrogen</td><td>10362-100</td><td>Alternatively, GeneJuice transfection (EMD) reagent can be used</td></tr><tr><td>Streptomycin sulphate (solid)</td><td>Melford</td><td>S0148</td><td>Sterilize filter (0.22 &amp;mu;m filter) before use</td></tr><tr><td>Penicillin G, potassium salt (solid)</td><td>Melford</td><td>P0580</td><td>Sterilize filter (0.22 &amp;mu;m filter) before use</td></tr><tr><td>CELLSTAR Sterile 6-well Culture Plate</td><td>Greiner Bio-One</td><td>657160</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>10100-147</td><td></td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>P8849</td><td>Caution: Protease inhibitors are dissolved in DMSO</td></tr><tr><td>Bovine pancrease deoxyribonuclease (Dnase) I&amp;nbsp;</td><td>Sigma</td><td>D5025</td><td></td></tr><tr><td>HisTrap FF (5 ml)</td><td>GE Heathcare</td><td>17-5286-01</td><td>Requires an &amp;Auml;kta FPLC machine</td></tr><tr><td>HiTrap Heparin (5 ml)</td><td>GE Heathcare</td><td>17-0407-01</td><td>Requires an &amp;Auml;kta FPLC machine</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units (with Ultracel-50 membrane)</td><td>Millipore</td><td>UFC905024</td><td>15 ml capacity and a MWCO of 50 kDa protein concentrator</td></tr></tbody></table>

efficient production and purification of recombinant murine kindlin-3 from insect cells for biophysical studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

The large scale expression of recombinant mouse kindlin-3 using baculovirus-infected Sf9 cells can take less than two weeks to achieve milligram quantities, as illustrated in the schematic Figure&#160;1A and requires only a small quantity of plasmid DNA from a QIAprep Miniprep kit, for example. The generation of recombinant baculovirus is achieved by cotransfecting Sf9 cells with mouse kindlin-3 (FERMT3)-containing plasmid together with an engineered linearized bacmid (BAC10:KO1629) and harvesting the newly formed virions after 5-7 days, as shown in Figure 1A. This method of baculovirus generation results in 100% recombinant viruses and essentially dispenses the need for plaque purification28,30. A representative small-scale (2 ml monolayer) culture will generate a recombinant virus-containing solution with an expected viral titer&#160;of 1 x 107 plaque forming units (pfu) per ml&#160;of culture. One can perform a plaque assay to determine the actual viral titer&#160;but this is perhaps too labor-intensive when a high number of constructs are tested for structural studies. The success of the virus generation step can be assessed by using an eGFP-containing plasmid in parallel, or, for this kindlin-3 construct, the Sf9 monolayer can be resuspended in PBS and assessed by SDS-PAGE and western blotting, which typically demonstrates a clear band corresponding to a 75 kDa His-tagged protein, as shown in Figure 1B. The virus is subsequently amplified to generate sufficient quantities for large-scale (liter&#160;volumes) insect cell infection and recombinant protein isolation. The second passage virus (P2) is generated by infecting suspension cultures with an estimated MOI of 0.1 (see protocol). It is vital that the amplification Sf9 suspension culture occupies only a twentieth of the total flask volume. This additional aeration ensures that the resulting virus-containing media, harvested after 3 days (72 hr) post-infection, will generate sufficient quantities of kindlin-3 in the subsequent expression culture. The amplified virus stock (P2) is assumed to possess an expected viral titer&#160;of 2 x 108 pfu/ml based on a conservative estimate of 100 pfu/cell using a cell density of 2 x 106&#160;cell/ml during the amplification31. Generally, for optimum baculovirus amplification and protein production, the Sf9 cells should be uniform in size and spherical, as shown in Figure&#160;1C. Additionally, viral amplification and protein expression is maximal at 72 hr&#160;post-infection, and both are significantly reduced at 96 hr&#160;post-infection.
Once virus has been amplified and used to infect Sf9 cells in large scale experiments recombinant kindlin-3 is partially purified by virtue of its engineered C-terminal His6-tag using immobilized metal affinity chromatography, as shown in Figure 2. It is important to carry out the purification at 4 &#176;C and that sufficient protease inhibitors have been added to prevent proteolysis. The partially-purified kindlin-3 is further purified to near homogeneity by ion exchange chromatography (IEC) using a heparin column, as shown in Figure&#160;3. We employed the use of a heparin column instead of a conventional ionic exchange column, as we predicted that the large number of basic residues, including a poly-lysine stretch within the kindlin-3 F1 domain, would interact strongly with the negatively charged sulphate groups of the column. This strategy is particularly useful for DNA- and RNA-binding proteins with basic nucleic acid-binding patches, for example the RNA-binding terminal uridylyltransferase Cid132.&#160;Finally kindlin-3 purity is &#34;polished&#34; by size exclusion chromatography to remove aggregates and achieve homogeneity, as shown in Figure 4. The buffers specified in the protocols are standard buffers frequently used in protein purification for structural analysis. Generally, phosphate-based buffers are avoided for the purification of proteins for structural studies, especially crystallization screening due to the formation of phosphate crystals in the crystallization drops (particularly for experiments at 4 &#176;C). However, we performed a Thermofluor-based thermal shift assay to determine which buffers were stabilizing for kindlin-3, as shown in Figure&#160;5. Briefly, a purified protein solution is diluted into buffers that cover a range of pH and sodium chloride concentrations, therefore forming a 2-dimensional screen. The melting of the protein is measured by observing the fluorescence from Sypro orange dye (molecular probes), that binds to hydrophobic residues within the folded protein core, over the temperature range 20-95 &#176;C (293-368 K). The temperature mid-point at which the protein unfolds (transition temperature, Tm) was calculated using the Opticon Monitor software and is described elsewhere33. Kindlin-3 was observed to be stable at high sodium chloride concentrations (500 mM) within the pH range 7.0-9.0, with a consistent transition temperature (Tm) of 55 &#176;C. It was also observed that the Tm of Kindlin-3 was approximately 55 &#176;C within the pH range 7.0-7.5 irrespective of sodium chloride concentration.
We found that there was limited proteolysis of kindlin-3 during the purification but SDS-PAGE analysis of highly concentrated protein (~15 mg/ml) demonstrated limited contamination with two additional polypeptides of equal intensity. Oddly, however there is no indication of additional species by size exclusion chromatography, as shown in Figure 4. It is thus thought that the protein is nicked by proteases but remains folded and hydrodynamically indistinguishable from full-length protein. Interestingly, Calpain is a known protease that cleaves kindlin-3 at Tyrosine373, which is in the &#946;1-&#946;2 loop of the pleckstrin homology (PH) domain34 and may explain our observations. Furthermore, western blotting reveals that one of the polypeptide doublets possesses a C-terminal His-tag and the apparent molecular weight of the doublet, when summed, equals 75 kDa,&#160;the same molecular weight as the native protein. The yield of purified recombinant kindlin-3 per liter&#160;of Sf9 cells (~2 g cell weight) is, at best, 5 mg.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig1highres.jpg" src="/files/ftp_upload/51206/51206fig1.jpg" /> 
Figure 1.&#160;Overview of baculovirus-infected insect cell heterologous protein expression. (A) A schematized overview of baculovirus generation and expression of kindlin-3 in Sf9 cells. In the DNA vector schematics, 5&#39;UTR/ORF603 is colored in red, the ORF1629 is colored in green and, the gene of the protein of interest (P.O.I.) is colored blue. (B) Western blot, using an anti-His6 antibody, of six small-scale (2 ml monolayer) cultures (lanes labelled according to culture) of Sf9 cells producing recombinant baculovirus and recombinant murine kindlin-3. (C) Light microscopy image of healthy Sf9 cells grown in suspension in Sf-900 II SFM supplemented with antibiotics and transferred to a 35&#160;mm well tissue culture dish (see protocol). Image is 20X&#160;magnification.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig2highres.jpg" src="/files/ftp_upload/51206/51206fig2.jpg" /> 
Figure 2.&#160;Representative purification of kindlin-3 by immobilized metal affinity chromatography (IMAC).&#160;SDS-PAGE of nickel affinity purified recombinant murine kindlin-3 expressed in baculovirus infected Sf9 cells (~2 g cell weight). Adsorbed protein was eluted using an imidazole gradient (shown above the gel). Lanes are labelled as follows; MW, molecular weight marker; Lys, whole cell lysate; FT, flow through (unbound); WI, wash 1; WII, wash 2. Western blot analysis (below) was also performed using the elution fractions to confirm the presence of the engineered His-tag on the recombinant protein. <a href="https://www.jove.com/files/ftp_upload/51206/51206fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig3highres.jpg" src="/files/ftp_upload/51206/51206fig3.jpg" /> 
Figure 3.&#160;Representative purification of kindlin-3 by Heparin Affinity Chromatography. (A) Elution profile observed at 280 nm (blue) displaying a single symmetrical peak eluting under a linear sodium chloride gradient (green) using the NaCl concentration range of 0.05-1.0 M. (B) SDS-PAGE analysis of the fractionated elution demonstrating the presence of the 75 kDa protein, kindlin-3 (labeled K3). <a href="https://www.jove.com/files/ftp_upload/51206/51206fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig4highres.jpg" src="/files/ftp_upload/51206/51206fig4.jpg" /> 
Figure 4.&#160;Representative gel filtration chromatography and concentrated kindlin-3. (A) Gel filtration elution profile of purified kindlin-3 using a Superdex S200 (16/60) in Tris-HCl, pH 7.5, 150 mM NaCl and 1 mM DTT at 20 &#176;C. Based on the elution volume, kindlin-3 migrates as expected for a 75 kDa protein, suggesting that it is predominantly monomeric. (B) SDS-PAGE of highly concentrated purified recombinant kindlin-3 at 14.5 mg/ml. <a href="https://www.jove.com/files/ftp_upload/51206/51206fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51206/51206fig5highres.jpg" src="/files/ftp_upload/51206/51206fig5.jpg" /> 
Figure 5.&#160;Thermofluor-based Thermal Shift Assay for Buffer Screening. Kindlin-3 was diluted into various buffers comprising a two-dimensional screen of pH versus sodium chloride concentration. Transition temperatures (temperature mid-points) were observed by fluorescence of the hydrophobically bound dye, Sypro orange (molecular probes) and calculated using the Opticon Monitor software. (A) A 3D histogram of the transition temperatures is plotted together with (B) the change in transition temperature from the calculated average of 50.4 &#176;C. For clarity the bars are colored according to the range of temperatures to which they correspond. <a href="https://www.jove.com/files/ftp_upload/51206/51206fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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Biology

23.3K Views.  University of Oxford. Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Video: Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Microcrystallography of Protein Crystals and In Cellulo Diffraction

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Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

Biochemistry

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Immunology and Infection

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

Neuroscience

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

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Chapters in this video

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys₂-His₂ Zinc-finger Domains

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization