We thank Prof. D. Madden, for many helpful discussions throughout this project. We thank Prof. D. Bolon for the gift of the plasmid containing the optimized GCN4 coiled coil. We thank Dr. B. Guo for NEMO plasmids. We thank Christina R. Arnoldy, Tamar Basiashvili and Amy E. Kennedy for demonstrating the procedure. We thank the BioMT Crystallography Core Facility and the departments of Chemistry and of Biochemistry &#38; Cell Biology at Dartmouth for the use of the crystallography equipment and the BioMT personnel for their support. This research used the AMX beamline of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. We thank the staff at NSLS II for their support. This work was funded by NIH grants R03AR066130, R01GM133844, R35GM128663 and P20GM113132, and a Munck-Pfefferkorn Novel and Interactive grant.

<ol>
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The authors declare no competing interests.

Crystallization attempts of NEMO in the unbound form were unsuccessful, including attempts using the full-length protein and several truncation constructs encompassing the IKK-binding domain. Our biophysical characterization of the IKK-binding domain of NEMO (residues 44-111) by circular dichroism, NMR spectroscopy and fluorescence anisotropy indicated that the construct, albeit able to bind IKK&#946;, existed in a state of conformational exchange, not suitable for crystallization9,<sup...

The NF-&#954;B pathway is activated in response to a variety of stimuli, including cytokines, microbial products and stress, to regulate expression of target genes responsible for inflammatory and immune response, cell death or survival and proliferation1. Pathologies including inflammatory and autoimmune diseases and cancer2,3,4,5 have been correlated to hyperactivation of the pathway, which has made modulation of NF-&#954;B activity a prime target for the development of new therapies6,7.
The canonical NF-&#954;B pathway in particular is distinguished from the non-canonical pathway, responsible for lymphorganogenesis and B-cell activation, by the former&#39;s dependence on the scaffolding protein NEMO (NF-&#954;B essential modulator8) for the assembly of the IKK-complex with the kinases IKK&#945; and IKK&#946;. The IKK complex is responsible for the phosphorylation of I&#954;B&#945; (inhibitor of &#954;B) that targets it for degradation, freeing the NF-&#954;B dimers to translocate to the nucleus for gene transcription1 and is therefore an attractive target for the development of inhibitors to modulate NF-&#954;B activity.
Our research focuses on the characterization of the protein-protein interaction between NEMO and IKK&#946;, targeting NEMO for the development of small molecules inhibitors of IKK complex formation. The minimal binding domain of NEMO, required to bind IKK&#946;, encompasses residues 44-111, and its structure has been determined in complex with a peptide corresponding to IKK&#946; sequence 701-7459. NEMO and IKK&#946; form a four-helix bundle where the NEMO dimer accommodates the two helices of IKK&#946;(701-745) in an elongated open groove with an extended interaction interface. IKK&#946;(734-742), also known as the NEMO-binding domain (NBD), defines the most important hot-spot for binding, where the two essential tryptophans (739,741) bury deeply within the NEMO pocket. The details of the complex structure can aid in the structure-based design and optimization of small molecule inhibitors targeting NEMO. At the same time, it is difficult that binding of a small molecule or peptide would recreate in NEMO the full conformational change (i.e., extensive opening of the NEMO coiled-coil dimer) caused by binding of the long IKK&#946;(701-745), as observed in the crystal, and the structure of unbound NEMO or NEMO bound to a small molecule inhibitor may represent a better target for structure-based drug design and inhibitor optimization.
Full length NEMO and smaller truncation constructs encompassing the IKK-binding domain have proven intractable for structure determination in the unbound form via X-ray crystallography and nuclear magnetic resonance (NMR) methods10, which prompted us to design an improved version of the IKK-binding domain of NEMO. Indeed, NEMO (44-111) in the unbound form is only partially folded and undergoes conformational exchange and we therefore set to stabilize its dimeric structure, coiled-coil fold and stability, while preserving binding affinity for IKK&#946;. By appending three heptads of ideal dimeric coiled-coil sequences11 at the N-and C-termini of the protein, and a series of four point mutations, we generated NEMO-EEAA, a construct fully dimeric and folded in a coiled coil, which rescued IKK-binding affinity to the nanomolar range as observed for full length NEMO12. As an additional advantage, we hoped the coiled-coil adaptors (based on the GCN4 sequence) would facilitate crystallization and eventually aid in the X-ray structure determination via molecular replacement. Coiled-coil adaptors have been similarly utilized to both increase stability, improve solution behavior and facilitate crystallization for trimeric coiled coils and antibody fragments13,14. NEMO-EEAA is easily expressed and purified from Escherichia. coli cells with a cleavable Histidine tag, is soluble, folded in a stable dimeric coiled coil and is easily crystallized, with diffraction to 1.9 &#197;. The presence of the ordered coiled-coil regions of GCN4 could additionally aid in phasing the data from crystals of NEMO-EEAA by molecular replacement using the known structure of GCN415.
Given the results obtained with apo-NEMO-EEAA, we believe the protocols described here could also be applied to the crystallization of NEMO-EEAA in the presence of small peptides (like the NBD peptide) or small molecule inhibitors, with the goal of understanding the requirements for NEMO inhibition and structure-based optimization of initial lead inhibitors to high affinity. Given the plasticity and dynamic nature of many coiled-coil domains16, the use of the coiled-coil adaptors could find more general applicability in aiding structural determination.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20% w/v &#947;-PGA (Na+ form, LM)</td>
			<td>Molecular Dimensions</td>
			<td>MD2-100-150</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>3.5 kDa MWCO Dialysis Membrane</td>
			<td>Spectra/Por</td>
			<td>132724</td>
			<td>For dialysis removal of imidazole</td>
		</tr>
		<tr>
			<td>Amicon Stirred Cell</td>
			<td>Millipose Sigma</td>
			<td>UFSC 05001</td>
			<td>For protein concentration</td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>Millipore Sigma</td>
			<td>G8270</td>
			<td>For minimal media labeling</td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Millipore Sigma</td>
			<td>9025-65-4</td>
			<td>For digestion of nucleic acid</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus (DE3)-RIL Competent Cells</td>
			<td>Agilent Technologies</td>
			<td>Model: 230245</td>
			<td>TEV expression</td>
		</tr>
		<tr>
			<td>CryoPro</td>
			<td>Hampton Research</td>
			<td>HR2-073</td>
			<td>Cryo-protectants kit</td>
		</tr>
		<tr>
			<td>D-Glucose (Dextrose)</td>
			<td>Millipore Sigma</td>
			<td>A9434</td>
			<td>For minimal media labeling</td>
		</tr>
		<tr>
			<td>Difco Terrific Broth</td>
			<td>ThermoFisher</td>
			<td>DF043817</td>
			<td>For culture growth</td>
		</tr>
		<tr>
			<td>Dithiothreitol &#62; 99%</td>
			<td>Goldbio</td>
			<td>DTT25</td>
			<td>For reduction of disulfides</td>
		</tr>
		<tr>
			<td>E. coli: Rosetta 2 (DE3)</td>
			<td>Novagen</td>
			<td>71400-3</td>
			<td>Expression of unlabeled NEMO-EEAA</td>
		</tr>
		<tr>
			<td>FORMULATOR</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Liquid handler/ screen builder</td>
		</tr>
		<tr>
			<td>HCl &#8211; 1.0 M Solution</td>
			<td>Hampton Research</td>
			<td>HR2-581</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HiLoad 16/600 Superdex 75 pg</td>
			<td>GE Healthcare</td>
			<td>28989333</td>
			<td>For size exclusion purification</td>
		</tr>
		<tr>
			<td>HisTrap HP 5 mL column</td>
			<td>GE Healthcare</td>
			<td>17524802</td>
			<td>For purification of His-tagged NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HT 96 MIDAS</td>
			<td>Molecular Dimensions</td>
			<td>MD1-59</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>HT 96 Morpheous</td>
			<td>Molecular Dimensions</td>
			<td>MD1-46</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>ThermoFisher</td>
			<td>288-32-4</td>
			<td>For elution from His-trap column</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside</td>
			<td>Goldbio</td>
			<td>I2481C5</td>
			<td>For induction of cultures</td>
		</tr>
		<tr>
			<td>MRC2 crystallization plate</td>
			<td>Hampton Research</td>
			<td>HR3-083</td>
			<td>Crystallization plate</td>
		</tr>
		<tr>
			<td>NT8 - Drop Setter</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Crystallization</td>
		</tr>
		<tr>
			<td>pET-16b</td>
			<td>Millipore Sigma</td>
			<td>69662</td>
			<td>For cloning of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>pET-45b</td>
			<td>Millipore Sigma</td>
			<td>71327</td>
			<td>For cloning of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride</td>
			<td>ThermoFisher</td>
			<td>36978</td>
			<td>For inhibition of proteases</td>
		</tr>
		<tr>
			<td>Polycarbonate Bottle for use in Ultracentrifuge Rotor Type 45 Ti</td>
			<td>Beckmann Coulter</td>
			<td>339160</td>
			<td>Ultracentrifuge bottle</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 20,000</td>
			<td>Hampton Research</td>
			<td>HR2-609</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>pRK793 (TEV)</td>
			<td>Addgene</td>
			<td>Plasmid 8827</td>
			<td>For TEV production</td>
		</tr>
		<tr>
			<td>QuikChange XL II</td>
			<td>Agilent Technologies</td>
			<td>200522</td>
			<td>Site directed mutagenesis</td>
		</tr>
		<tr>
			<td>Required Cap Assembly:</td>
			<td>Beckmann Coulter</td>
			<td>355623</td>
			<td>Ultracenttrifuge bottle cap</td>
		</tr>
		<tr>
			<td>ROCK IMAGER</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Crystallization Imager</td>
		</tr>
		<tr>
			<td>Seed Bead Kit</td>
			<td>Hampton Research</td>
			<td>HR2-320</td>
			<td>Seed generation</td>
		</tr>
		<tr>
			<td>Sodium Chloride &#8805; 99%</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td>For buffering of purification solutions</td>
		</tr>
		<tr>
			<td>TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride)</td>
			<td>Goldbio</td>
			<td>TCEP1</td>
			<td>Reducing agent</td>
		</tr>
		<tr>
			<td>The Berkeley Screen</td>
			<td>Rigaku</td>
			<td>MD15-Berekely</td>
			<td>For sparse matrix screening of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>The PGA Screen</td>
			<td>Molecular Dimensions</td>
			<td>MD1-50</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Tris &#8211; 1.0 M Solution</td>
			<td>Hampton Research</td>
			<td>HR2-589</td>
			<td>For fine screen crystallization of NEMO-EEAA</td>
		</tr>
		<tr>
			<td>Ultrapure Tris Buffer (powder format)</td>
			<td>Thermofisher</td>
			<td>15504020</td>
			<td>For buffering of purification solutions</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>ThermoFisher</td>
			<td>29700</td>
			<td>For denaturation of NEMO-EEAA</td>
		</tr>
	</tbody>
</table>

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.

Cloning, expression and purification of the IKK-binding domain of NEMO. 
The protocol followed in this study to obtain the final sequence of NEMO-EEAA (Figure 1A), which produced diffraction quality crystals, involved the expression and characterization of all the intermediate constructs, including the addition of the coiled-coil adaptors at N- and or C-terminus, the mutations C76A, C95S and the mutations E56A, E57A. Figure 1</stron...

Watch this Scientific Journal Video about Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO at JoVE.com

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

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 ...

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7.4K Views.  Dartmouth College. 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.

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

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

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 ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation.
Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments&#39; ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

We describe here protocols for the measurement of antibody-antigen binding affinity and kinetics using four commonly used biosensor platforms.

Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

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.

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 ...

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. These features, among others, make single domain antibodies an invaluable tool for biomedical research. A method for the production of single domain antibodies from alpacas is reported. A protocol for immunization, blood collection, and B-cell isolation is described. The B-cells are used for the construction of an immunized library, which is used in the selection of specific single domain antibodies via panning. Putative specific single domain antibodies obtained via panning are confirmed by pull-down, ELISA, or gel-shift assays. The resulting single domain antibodies can then be used either directly or as a part of an engineered reagent. The uses of single domain antibody and single domain antibody-based regents include structural, biochemical, cellular, in vivo, and therapeutic applications. Single domain antibodies can be produced in large quantities as recombinant proteins in prokaryotic expression systems, purified, and used directly or can be engineered to contain specific markers or tags that can be used as reporters in cellular studies or in diagnostics.

Immunization of Alpacas Lama pacos with Protein Antigens and Production of Antigen-specific Single Domain Antibodies

A method for the production of single domain antibodies from alpacas, including immunization, blood collection, B-cell isolation, and selection is described.

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

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 ...