Name: production of human norovirus protruding domains in e. coli for x-ray crystallography
Uploaded: 2016-04-19T14:00:00
Description: Watch this Scientific Journal Video about Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography at JoVE.com

The funding for this study was provided by the CHS foundation. We acknowledge the protein crystallization platform within the excellence cluster CellNetworks of the University of Heidelberg for crystal screening and the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities.

1. P Domain Cloning
<ol>
	<li>Determine the P domain coding region by sequence alignment of norovirus strains (e.g., GII.10 strain, GenBank: AF504671, pdb-ID: 3ONU)6. Moreover, remove the flexible region at the C-terminal end of the P domain (Figure 2A). Codon-optimize the DNA for E. coli expression and include BamHI (N-terminal) and NotI (C-terminal) restriction sites in order to sub-clone the P domain coding region into the pMalc2x expression vector6,7. 
	...

     

<ol>
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K., 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=Enteric+bacteria+promote+human+and+mouse+norovirus+infection+of+B+cells.">Enteric bacteria promote human and mouse norovirus infection of B cells.</a> Science. 346, 755-759 (2014).</li><li>Hansman, G. S., 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=Crystal+structures+of+GII.10+and+GII.12+norovirus+protruding+domains+in+complex+with+histo-blood+group+antigens+reveal+details+for+a+potential+site+of+vulnerability.">Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability.</a> J. Virol. 85, 6687-6701 (2011).</li><li>Fath, S., 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=Multiparameter+RNA+and+codon+optimization:+a+standardized+tool+to+assess+and+enhance+autologous+mammalian+gene+expression.">Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.</a> PLoS One. 6 (e17596), (2011).</li><li>Jacob, F., Monod, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulatory+mechanisms+in+the+synthesis+of+proteins.">Genetic regulatory mechanisms in the synthesis of proteins.</a> J. Mol. Biol. 3, 318-356 (1961).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Processing+of+Rotation+Diffraction+Data+from+Crystals+of+Initially+Unknown+Symmetry+and+Cell+Constants.">Automatic Processing of Rotation Diffraction Data from Crystals of Initially Unknown Symmetry and Cell Constants.</a> J. Appl. Crystallogr. 26, 795-800 (1993).</li><li>Mccoy, A. 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=Phaser+crystallographic+software.">Phaser crystallographic software.</a> J. Appl. Crystallogr. 40, 658-674 (2007).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallogr. Sect. D-Biol. Crystallogr. 66, 486-501 (2010).</li><li>Adams, P. D., 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=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallogr. Sect. D-Biol. Crystallogr. 66, 213-221 (2010).</li><li>Koromyslova, A. D., Hansman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobody+binding+to+a+conserved+epitope+promotes+norovirus+particle+disassembly.">Nanobody binding to a conserved epitope promotes norovirus particle disassembly.</a> J. Virol. 89, 2718-2730 (2015).</li><li>Hansman, G. S., 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=Structural+basis+for+broad+detection+of+genogroup+II+noroviruses+by+a+monoclonal+antibody+that+binds+to+a+site+occluded+in+the+viral+particle.">Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle.</a> J. Virol. 86, 3635-3646 (2012).</li><li>Singh, B. K., Leuthold, M. M., Hansman, G. 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Here, we describe a protocol for the expression and purification of norovirus P domains in high quality and quantity. Noroviruses are not well studied and structural data are continuously needed. To our knowledge, P domain production using other protocols (e.g., GST-tagged P domains) has been problematic, so far, and sufficient structural data on norovirus-host interaction have been missing. With the method described here, we have recently contributed significantly to the understanding of the molecular details o...

Human noroviruses are the major cause of acute gastroenteritis worldwide1. These viruses belong to the Caliciviridae family, of which there are at least five genera, including Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus. Despite their high impact on the healthcare system and wide distribution, the study of human noroviruses is hampered by the lack of a robust cell culture system. To date, there are no approved vaccines or antiviral strategies available.
The norovirus major capsid protein, termed VP1, can be divided into a shell (S) domain and a protruding (P) domain2. The P domain is connected to the S domain by a flexible hinge (H) region. The S domain forms a scaffold around the viral RNA, whereas the P domain forms the outmost part of the viral capsid. The P domain assembles into biologically relevant dimers when expressed in bacteria. The P dimer interacts with carbohydrate structures, termed histo-blood group antigens (HBGAs) that are present as soluble antigens in saliva and found on certain host cells3. The P domain-HBGA interaction is thought to be important for infection4. Indeed, a recent report revealed the importance of synthetic HBGAs or HBGA-expressing bacteria for human norovirus infection in vitro5.
Current studies regarding the host cell attachment of noroviruses are mainly performed with virus-like particles (VLPs) that can be expressed in insect cells or with recombinant P domains expressed in Escherichia coli (E. coli). To understand the P domain-HBGA interactions at atomic resolution, P domain-HBGA complex structures can be solved using X-ray crystallography. Here, we describe a protocol for P domain expression and purification that allows production of P domain in high quantity and quality to be used for X-ray crystallography. Moreover, this method can be applied for other calicivirus P domains and non-structural proteins.
The P domain is codon-optimized for E. coli expression and cloned into a standard transfer vector. The P domain is then re-cloned into an expression vector that encodes a polyhistidine (His) tag and a mannose-binding protein (MBP) that are followed by a protease cleavage site. The MBP-His-P domain fusion protein is expressed in E. coli, followed by three purification steps. The MBP-His-P domain fusion protein is purified using immobilized metal ion affinity chromatography (IMAC). Next, the fusion protein is cleaved with human rhinovirus (HRV)-3C protease and the P domain is separated from the MBP-His by an additional IMAC purification step. Lastly, the P domain is purified using size exclusion chromatography (SEC). The purified P domain can then be used for X-ray crystallography. Screening of protein crystallization conditions is performed with commercially available screening kits using different P domain protein concentrations. Crystal growth is observed and the most promising conditions are optimized.
With the methods described here, it is possible to go from gene to protein to structure within less than four weeks. Therefore, our method of P domain expression, purification, and crystallization is suitable to study norovirus-host interaction at the molecular level and provide important data to assist in up-to-date vaccine design and drug screening.

<table>
	<tbody>
		<tr>
			<td>P domain DNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>GeneArt Gene Synthesis</td>
		</tr>
		<tr>
			<td>pMalc2x&#160; vector</td>
			<td>On request</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136L</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>New England Biolabs</td>
			<td>R0189L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C. Medium</td>
			<td>Life Technologies</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Econo-Column Chromatography Column</td>
			<td>Bio-Rad</td>
			<td>7372512</td>
			<td>2.5 cm x 10 cm, possible to use other size</td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Vivaspin 20</td>
			<td>GE Healthcare</td>
			<td>various</td>
			<td>cutoff of 10 kDa, 30 kDa and 50 kDa used</td>
		</tr>
		<tr>
			<td>Subcloning Efficiency DH5&#945; Competent Cells</td>
			<td>Life Technologies</td>
			<td>18265-017</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot BL21(DE3) Chemically Competent E. coli</td>
			<td>Life Technologies</td>
			<td>C6000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HRV 3C Protease</td>
			<td>Merck Millipore</td>
			<td>71493</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 26/600 Superdex 75 PG</td>
			<td>GE Healthcare</td>
			<td>28-9893-34</td>
			<td>SEC column</td>
		</tr>
		<tr>
			<td>JCSG Core suites</td>
			<td>Qiagen</td>
			<td>various</td>
			<td>4 screens with each 96 wells</td>
		</tr>
		<tr>
			<td>Carbohydrates</td>
			<td>Dextra Laboratories, UK</td>
			<td>various</td>
			<td>Blood group products</td>
		</tr>
	</tbody>
</table>

production of human norovirus protruding domains in e. coli for x-ray crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

The schematic of the described protocol is depicted in Figure 1. The protocol covers 6 major parts that include cloning of the target gene, expression, a three-step purification, and crystallization. Figure 2 illustrates the design of the expression construct (EC) and characteristics of the pMalc2x expression vector. The sequence of the multiple cloning site (MCS) of the pMalc2x vector shows restriction and protease cleavage sites. Figure 3</stron...

Watch this Scientific Journal Video about Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography at JoVE.com

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

The overall goal of the protocol is the production and purification of human norovirus protruding domain in E.coli in order to obtain extremely pure protein for X-ray crystallography. Our protocol for protein expression, purification, and crystallization is fast, robust, and can be used for structural and nonstructural proteins. The advantage of this protocol is that our proteins are very pure and can form crystals that can diffract to high resolution.According to our experience, the protocol might need adjustment for less soluble proteins. For example, the growth medium or growth temperature during protein expression can be changed. To begin, transform one microliter of the pMalc2x vector, coding for the MBP-His-P domain fusion protein into 15 microliters of competent E.coli BL21 cells using a standard transformation protocol.Add 600 microliters of SOC medium to the cells. And grow for one hour at 37 degrees Celsius. Subculture the transformed cells into 120 milliliters of LB-ampicillin overnight at 160 rpm and 37 degrees Celsius.Inoculate nine liters of LB-ampicillin with the subculture. Grow the cells in the incubator at 37 degrees Celsius with a shaking speed of 160 rpm until the optical density at 600 nanometers reaches 0.4 to 0.6. Subsequently, lower the temperature to 22 degrees Celsius for approximately one hour.It is important to wait until the temperature reaches 22 degrees Celsius before inducing with a low amount of IPTG to allow for soluble protein expression. Induce the protein expression with 0.66 millimolar of IPTG. Following induction, grow the cells overnight at 22 degrees Celsius.Harvest the cells by centrifugation. Discard the supernatant and freeze the cell pellet at 20 degrees Celsius. Prepare buffers that are used for the protein purification steps from stock solutions as detailed in the text protocol.Thaw the cell pellet from the nine liter culture and dissolve in 150 milliliters of phosphate buffered saline at four degrees Celsius. Sonicate the cell suspension three times for two minutes to disrupt the cells. Centrifuge the sonicated cell suspension to separate cell debris from the supernatant containing expressed MBP-His-P domain fusion protein.Collect the supernatant and discard the pellet. Wash and equilibrate a 10-milliliter slurry of nickel-NTA agarose beads with 10 millimolar imidazole buffer in a chromatography column. Add the equilibrated nickel beads to the supernatant containing expressed MBP-His-P domain fusion protein and incubate for 30 minutes at four degrees Celsius with slow rotation.After incubation, apply the entire nickel bead-protein mixture to a chromatography column. Wash the column slowly with five CVs each of 10 millimolar, 20 millimolar, and 50 millimolar imidazole buffers at four degrees Celsius. Elute the MBP-His-P domain fusion protein using 250 millimolar imidazole buffer at four degrees Celsius.During elution, check the optical density at 280 nanometers to verify the elution of the fusion protein. Continue the elution until the optical density at 280 nanometers drops to approximately 0.1. Working at four degrees Celsius, wash the beads with excessive amount of 250 millimolar imidazole buffer, followed by 10 millimolar imidazole buffer.Save the beads for the second purification step. Verify the presence of the MBP-His-P domain fusion protein with sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, using a 12%SDS polyacrylamide gel. Then concentrate the eluted MBP-His-P domain fusion protein to a final concentration of approximately three milligrams per milliliter.The amount of protease needed to effectively cleave the MBP-His-P domain needs to be carefully calculated. It depends on the total amount of eluted fusion protein and on SDS-PAGE result. Cleave the MBP-His-P domain fusion with HRV 3C protease during dialysis against two liters of 10 millimolar imidazole buffer overnight at four degrees Celsius.Depending on the final volume of concentrated protein, perform dialysis in a dialysis cassette or dialysis tubing. Equilibrate the nickel beads that were set aside in 10 millilmolar imidazole buffer. Incubate the dialyzed protein containing the cleaved P domain, MBP protein, and HRV protease with the equilibrated nickel beads for 30 minutes at four degrees Celsius with slow rotation.Apply the nickel bead mixture to a column and collect the flowthrough at four degrees Celsius. This is the cleaved P domain. Measure the concentration of protein as it comes off the column until the optical density at 280 nanometers reaches approximately 0.1.The MBP-His should remain bound to the nickel beads and the cleaved P domain is eluted in the flowthrough. Check the presence of cleaved P domain using SDS-PAGE with a 12%gel as before. After concentrating the eluted P domain to approximately three milligrams per milliliter, dialyze overnight at four degrees Celsius against gel filtration buffer for subsequent size exclusion chromatography purification.Wash the pumps and pipes of the high performance liquid chromatography purification system, and pre-equilibrate the size exclusion chromatography column with gel filtration buffer. Inject the P domain onto the column at a flow rate of one milliliter per minute, using a superloop or loop, depending on the volume of the concentrated sample. After the injection is finished, increase the flow rate to 2.5 milliliters per minute.As the optical density increases, and the P domain comes off the column, collect fractions of 1.5 milliliters. The P domain is usually eluted as a dimer. After checking the fractions using SDS-PAGE with a 12%gel, pool only the purest fractions and concentrate to approximately three milligrams per milliliter and approximately eight milligrams per milliliter.Use the P domain at approximately three milligrams per milliliter and eight milligrams per milliliter for initial crystallization screening. Prepare at least 100 microliters of P domain per concentration for initial screens with 384 commercially available screening conditions. Perform screening at 18 degrees Celsius in a 96-well plate format where the reservoir contains 100 microliters of mother solution and a drop is composed of 0.2 microliters of mother solution and 0.2 microliters of protein.Optimize the successful crystallization conditions using 15-well plates that contain three rows. Use 500 microliters of mother solution as a reservoir volume. Set up the first row with 100%mother solution.The second row with 90%mother solution and 10%water. And the third row with 80%mother solution and 20%water. Set up the hanging drop with a drop size of two microliters by mixing an equal volume of protein and mother solution.Use the optimized crystal conditions for co-crystallizing the P domain with ligands. Prepare plates as before but instead of a two microliter drop size, set up drops containing one microliter of mother solution, one microliter of protein, and one microliter of ligand at a concentration of one milligram per milliliter. Shown here is a representative SDS-PAGE result, showing the amount of eluted MBP-His-P domain fusion protein after the first purification step, where the MBP-His-P domain fusion protein is purified using nickel-NTA agarose beads.Following separation of the MBP-His from the cleaved P domain, the amount of eluted cleaved P domain is also analyzed with SDS-PAGE. After the final purification step with size exclusion chromatography, crystal screening resulted in optimized crystal conditions. Here, the diffraction pattern of a protruding domain is shown.In principle, it is possible to clone, expressed, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains. While attempting this procedure, it is important to remember that quality is more important than quantity. Owing to the high quality of the purified P domains, additional studies are suitable, including immunization for antibody production, NMR experiments, and ELISA-based studies.Moreover, the purified P domain can be used for complex formation with antibody fragments and nanobodies. After watching this protocol, you should have a good understanding of how to express and purify norovirus P domains of high quality.

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