We thank the staff at the beamlines 19ID, 24ID, and 13ID at Advanced Photon Source, Lemont, IL, for support during data collection on various crystals. We are grateful to Dmitry Lyumkis, Salk Institute for fetching us the low resolution cryo-EM map at early stages of EM map/model building, which was used to build the initial IKK1 molecular replacement search model. The research leading to these results has received funding from NIH grants AI064326, CA141722, and GM071862 to GG. SP is currently a Wellcome Trust DBT India Intermediate Fellow.

<ol>
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The authors declare no competing financial interests or other conflicts of interest.

Production, crystallization and structure solution of two related IKK proteins 
We set out to determine the X-ray crystal structure of IKK1/&#945; with the notion that it would be a relatively straightforward exercise given our experience with IKK2/&#946; protein production, crystallization, and structure determination. However, we were highly surprised that these two related proteins behaved very differently regarding the ease of crystallization. Despite efforts from several high-profile laboratori...

Transcriptional activities of the NF-&#954;B family of dimeric transcription factors are required for diverse cellular functions ranging from inflammation and immunity to survival and death. These activities are stringently controlled in cells and a loss of regulation leads to various pathological conditions, including autoimmune disorders, and cancer1,2,3. In the absence of a stimulus, the activities of NF-&#954;B are kept inhibited by I&#954;B (Inhibitor of -&#954;B) proteins4. The phosphorylation of specific Ser residues on I&#954;B proteins marks them for ubiquitination and subsequent proteasomal degradation or selective processing5. Two highly homologous Ser/Thr kinases, IKK2/&#946; and IKK1/&#945;, act as central regulators of NF-&#954;B activities by carrying out these phosphorylation events6,7.
Interactions between a ligand and a receptor transduces a signal through a series of mediators leading to the activation of NF-&#954;B factors. The NF-&#954;B signalling process can broadly be classified into two distinct pathways &#8211; canonical and non-canonical (alternative)8. The activity of IKK2/&#946; primarily regulates the NF-&#954;B signalling of the canonical pathway that is essential for inflammatory and innate immune responses9. A distinct feature of this pathway is a rapid and short-lived activation of IKK2/&#946;10 within a hitherto biochemically uncharacterized IKK complex &#8212; presumed to be composed of IKK1 and IKK2, as well as a regulatory component, NEMO (NF-&#954;B Essential Modulator)11,12,13. Between the two catalytic IKK subunits of the IKK complex, IKK2 is primarily responsible14 for the phosphorylation of specific residues of prototypical I&#954;Bs (&#945;, -&#946;, &#38; -&#947;) bound to NF-&#954;B, and also an atypical I&#954;B protein, NF-&#954;B1/p105, which is a precursor of the NF-&#954;B p50 subunit5. Phosphorylation induced ubiquitination and proteasomal degradation of I&#954;B (or processing of p105) leads to the release and activation of a specific set of NF-&#954;B dimers15. Aberrant NF-&#954;B activity due to mis-regulated function of IKK2 has been observed in many cancers as well as in autoimmune disorders2,3,16.
In contrast to IKK2/&#946;, activity of IKK1/&#945; regulates NF-&#954;B signalling of the non-canonical pathway, which is essential for development and immunity. IKK1 phosphorylates specific residues of NF-&#954;B2/p100 on its C-terminal I&#954;B&#948; segment, which leads to its processing and the generation of p52. The formation of transcriptionally active p52:RelB heterodimer initiates a slow and sustained response to developmental signals7,17,18,19,20. Interestingly, the generation of the central NF-&#954;B factor p52 of this pathway is critically dependent on another factor, NF-&#954;B Inducing Kinase (NIK)21,22, but not on IKK2 or NEMO. In resting cells, the level of NIK remains low due to its constant proteasome-dependent degradation23,24,25. Upon stimulation of cells by &#39;non-canonical&#39; ligands, and in certain malignant cells, NIK becomes stabilized to recruit and activate IKK1/&#945;. Kinase activities of both NIK and IKK1 are essential for efficient processing of p100 into p527. IKK1 and NIK phosphorylate three serines (Ser866, 870 and 872) of NF-&#954;B2/p100 on its C-terminal I&#954;B&#948; segment leading to its processing and the generation of p52. Aberrant activation of the non-canonical pathway has been implicated in many malignancies including multiple myeloma26,27,28.
Several highly efficient and specific inhibitors for IKK2/&#946; are known, although none so far have turned out to be an effective drug. In contrast, IKK1/&#945;-specific inhibitors are sparse. This may stem partly from our lack of structural and biochemical information on IKK1/&#945;, which limits our understanding of the mechanistic basis of activation of NF-&#954;B by IKK1 in cells, and rational drug design. The X-ray structures of IKK2/&#946; provided us with insights into the activation mechanism of IKK2/&#946;29; however, these structures could not reveal how different upstream stimuli trigger activation of IKK1/&#945; or IKK2/&#946; to regulate distinct sets of NF-&#954;B activities 30,31. To understand the mechanistic basis underlying the distinct signalling function of IKK1/&#945;, and to establish a platform for rational drug design, we focused on determining the structure of IKK1/&#945;.

<table><tbody><tr><td>Cellfectin/Cellfectin II</td><td>Thermo Fisher Scientific</td><td>10362100</td><td>Cellfectin is now discontinued, replaced by Cellfectin II</td></tr><tr><td>Sf900 III Insect cell medium</td><td>Thermo Fisher Scientific</td><td>12658-027</td><td></td></tr><tr><td>SF9 cells</td><td>Thermo Fisher Scientific</td><td>12659017</td><td></td></tr><tr><td>anti-IKK1 antibody</td><td>Novus Biologicals</td><td>NB100-56704</td><td>Previously sold by Imgenex</td></tr><tr><td>anti-PentaHis antibody</td><td>Qiagen</td><td>34460</td><td></td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>IPVH00010</td><td>Nitrocellulose can also be used</td></tr><tr><td>Ni-NTA agarose</td><td>Qiagen</td><td>30210</td><td></td></tr><tr><td>Bradford assay reagent</td><td>BioRad</td><td>500001</td><td></td></tr><tr><td>Superdex 200 column</td><td>GE Healthcare</td><td>28989335</td><td></td></tr><tr><td>Amicon concentrator</td><td>Millipore</td><td>UFC801008, UFC803008, UFC201024, UFC203024</td><td></td></tr><tr><td>Compound A</td><td>Bayer</td><td></td><td></td></tr><tr><td>Calbiochem IKK-inhibitor XII</td><td>Calbiochem</td><td>401491</td><td></td></tr><tr><td>Staurosporine</td><td>SIGMA</td><td>S4400</td><td></td></tr><tr><td>MLN120B</td><td>Millenium</td><td>Gift item</td><td></td></tr><tr><td>AMPPNP</td><td>SIGMA</td><td>A2647</td><td></td></tr><tr><td>Dextran sulfate</td><td>SIGMA</td><td>51227, 42867, 31404,</td><td></td></tr><tr><td>Dextran sulfate</td><td>Alfa Aesar</td><td>J62101</td><td></td></tr><tr><td>PEG</td><td>SIGMA</td><td>93593, 81210, 88276, 95904, 81255, 89510, 92897, 81285, 95172</td><td>Some of them are new Cat # on SIGMA catalogue. What we had was originally from Fluka that had different Cat #.</td></tr><tr><td>Crystallization Screens</td><td></td><td></td><td></td></tr><tr><td>Crystal Screen I and II (Crystal Screen HT)</td><td>Hampton Research</td><td>HR2-130</td><td></td></tr><tr><td>Index HT</td><td>Hampton Research</td><td>HR2-134</td><td></td></tr><tr><td>PEG/Ion and PEG/Ion2 (PEG/Ion HT)</td><td>Hampton Research</td><td>HR2-139</td><td></td></tr><tr><td>PEGRX 1 and PEGRx 2 (PEGRx HT)</td><td>Hampton Research</td><td>HR2-086</td><td></td></tr><tr><td>SaltRx 1 and SaltRx 2 (SaltRx HT)</td><td>Hampton Research</td><td>HR2-136</td><td></td></tr><tr><td>Crystal mounts</td><td>Hampton Research</td><td>HR8-188, 190, 192, 194</td><td></td></tr><tr><td>Synchrotron</td><td>The Advanced Photon Source (APS) at the U.S. Department of Energy&amp;rsquo;s Argonne National Laboratory</td><td>Beamline 19 ID</td><td>The Advanced Photon Source (APS) at the U.S. Department of Energy&amp;rsquo;s Argonne National Laboratory provides ultra-bright, high-energy storage ring-generated X-ray beams for research in almost all scientific disciplines.</td></tr></tbody></table>

a guide to production, crystallization, and structure determination of human ikk1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

Cloning and expression of different constructs of IKK1/&#945; 
Full length human IKK1/&#945; was cloned into the baculovirus expression vector pFastBacHTa within its EcoRI and NotI restriction sites to obtain an N-terminal hexa-Histidine tagged IKK1. The tag could be removed by TEV protease digestion. Since full length IKK1/&#945; contains flexible regions on both ends, and flexible regions usually render a protein difficult to crystallize, we cloned various truncate...

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A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

a-guide-to-production-crystallization-structure-determination-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.1K Views.  University of California, San Diego. IκB Kinase 1/α (IKK1/α CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-κB transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

Video: A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

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 ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous &#946;2m is not available. Meanwhile, it is indicated that mammalian &#946;2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m). Herein, methods to evaluate the feasibility of heterologous &#946;2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &#946;2-Microglobulin

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential &#946;2-microglobulin (&#946;2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous &#946;2m were investigated.

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...

Immunology and Infection

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.

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 ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α

Summary

Explore More Videos

Chapters in this video

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous β₂-Microglobulin

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