Name: fully processed recombinant kras4b: isolating and characterizing the farnesylated and methylated protein
Uploaded: 2020-01-16T14:00:00
Description: Watch this Scientific Journal Video about Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein at JoVE.com

We acknowledge cloning and expression support from Carissa Grose, Jen Melhalko, and Matt Drew in the Protein Expression Laboratory, Frederick National Laboratory for Cancer Research. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government

1. Protein purification
<ol>
	<li>Prepare buffers A&#8211;H, as seen in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer solution</td>
			<td>Buffering agent (all 20 mM)</td>
			<td>pH</td>
			<td>NaCl (mM)</td>
			<td>imidazole (mM)</td>
			<td>MgCl2</td>
			<td>TCE<...

<ol>
	<li>Cox, A. D., Der, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+prenylation:+more+than+just+glue.">Protein prenylation: more than just glue.</a> Current Opinion in Cell Biology. 4 (6), 1008-1016 (1992).</li><li>Zhang, F. L., Casey, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Prenylation:+Molecular+Mechanisms+and+Functional+Consequences.">Protein Prenylation: Molecular Mechanisms and Functional Consequences.</a> Annual Review of Biochemistry. 65, 241-269 (1996).</li><li>Hottman, D. A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+prenylation+and+synaptic+plasticity:+implications+for+Alzheimer's+disease.">Protein prenylation and synaptic plasticity: implications for Alzheimer's disease.</a> Molecular Neurobiology. 50 (1), 177-185 (2014).</li><li>Hottman, D. A., Chernick, D., Cheng, S., Wang, Z., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL+and+cognition+in+neurodegenerative+disorders.">HDL and cognition in neurodegenerative disorders.</a> Neurobiology of Disease. 72, 22-36 (2014).</li><li>Nakagami, H., Jensen, K. S., Liao, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+pleiotropic+effect+of+statins:+prevention+of+cardiac+hypertrophy+by+cholesterol-independent+mechanisms.">A novel pleiotropic effect of statins: prevention of cardiac hypertrophy by cholesterol-independent mechanisms.</a> Annals of Medicine. 35 (6), 398-403 (2003).</li><li>Kohnke, M., 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=Rab+GTPase+prenylation+hierarchy+and+its+potential+role+in+choroideremia+disease.">Rab GTPase prenylation hierarchy and its potential role in choroideremia disease.</a> PLoS One. 8 (12), 81758 (2013).</li><li>Berndt, N., Hamilton, A. D., Sebti, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+protein+prenylation+for+cancer+therapy.">Targeting protein prenylation for cancer therapy.</a> Nature Reviews Cancer. 11 (11), 775-791 (2011).</li><li>Kho, 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=A+tagging-via-substrate+technology+for+detection+and+proteomics+of+farnesylated+proteins.">A tagging-via-substrate technology for detection and proteomics of farnesylated proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (34), 12479-12484 (2004).</li><li>Armstrong, S. A., Hannah, V. C., Goldstein, J. L., Brown, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAAX+geranylgeranyl+transferase+transfers+farnesyl+as+efficiently+as+geranylgeranyl+to+RhoB.">CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB.</a> Journal of Biological Chemistry. 270 (14), 7864-7868 (1995).</li><li>Stephen, A. G., Esposito, D., Bagni, R. K., McCormick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dragging+ras+back+in+the+ring.">Dragging ras back in the ring.</a> Cancer Cell. 25 (3), 272-281 (2014).</li><li>Denisov, I. G., Sligar, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanodiscs+in+Membrane+Biochemistry+and+Biophysics.">Nanodiscs in Membrane Biochemistry and Biophysics.</a> Chemical Reviews. 117 (6), 4669-4713 (2017).</li><li>Bao, H., Duong, F., Chan, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Step-by-step+Method+for+the+Reconstitution+of+an+ABC+Transporter+into+Nanodisc+Lipid+Particles.">A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles.</a> Journal of Visualized Experiments. (66), e3910 (2012).</li><li>Dementiev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K-Ras4B+lipoprotein+synthesis:+biochemical+characterization,+functional+properties,+and+dimer+formation.">K-Ras4B lipoprotein synthesis: biochemical characterization, functional properties, and dimer formation.</a> Protein Expression and Purification. 84 (1), 86-93 (2012).</li><li>Lowe, P. N., 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=Characterization+of+recombinant+human+Kirsten-ras+(4B)+p21+produced+at+high+levels+in+Escherichia+coli+and+insect+baculovirus+expression+systems.">Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems.</a> Journal of Biological Chemistry. 266 (3), 1672-1678 (1991).</li><li>Gillette, W., 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=Production+of+Farnesylated+and+Methylated+Proteins+in+an+Engineered+Insect+Cell+System.">Production of Farnesylated and Methylated Proteins in an Engineered Insect Cell System.</a> Methods in Molecular Biology. 2009, 259-277 (2019).</li><li>Agamasu, C., 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=KRAS+Prenylation+Is+Required+for+Bivalent+Binding+with+Calmodulin+in+a+Nucleotide-Independent+Manner.">KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in a Nucleotide-Independent Manner.</a> Biophysical Journal. 116 (6), 1049-1063 (2019).</li><li>Talsania, 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=Genome+Assembly+and+Annotation+of+the+Trichoplusia+ni+Tni-FNL+Insect+Cell+Line+Enabled+by+Long-Read+Technologies.">Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies.</a> Genes. 10 (2), 79 (2019).</li><li>Spencer-Smith, R., 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=Inhibition+of+RAS+function+through+targeting+an+allosteric+regulatory+site.">Inhibition of RAS function through targeting an allosteric regulatory site.</a> Nature Chemical Biology. 13 (1), 62-68 (2016).</li><li>Lowe, P. N., 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=Expression+of+polyisoprenylated+Ras+proteins+in+the+insect/baculovirus+system.">Expression of polyisoprenylated Ras proteins in the insect/baculovirus system.</a> Biochemical Society Transactions. 20 (2), 484-487 (1992).</li><li>Dharmaiaha, 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+of+recognition+of+farnesylated+and+methylated+KRAS4b+by+PDE&#948;.">Structural basis of recognition of farnesylated and methylated KRAS4b by PDE&#948;.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (44), 6766-6775 (2016).</li><li>Abdiche, Y. N., Myszka, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+mechanism+of+drug/Lipid+membrane+interactions+using+Biacore.">Probing the mechanism of drug/Lipid membrane interactions using Biacore.</a> Analytical Biochemistry. 328 (2), 233-243 (2004).</li><li>Fisher, R. 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=Complex+interactions+of+HIV-1+nucleocapsid+protein+with+oligonucleotides.">Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides.</a> Nucleic Acids Research. 34 (2), 472-484 (2006).</li><li>Lakshman, B., 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=Quantitative+biophysical+analysis+defines+key+components+modulating+recruitment+of+the+GTPase+KRAS+to+the+plasma+membrane.">Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane.</a> Journal of Biological Chemistry. 294, 2193-2207 (2019).</li></ol>

As noted in the Representative Results section, the most critical step during the purification is the handling of the sample during the time it is in lower salt. Limiting the time that the sample is exposed to less than 200 mM NaCl will help reduce precipitation and increase sample yield. Interpretation of the results of the CEX can be difficult if the profile does not match the expectations (see Figure 2). Until the protocol has become routine, it is advised that the CEX elution fractions t...

Posttranslational modifications play a key role in defining the functional activity of proteins. Modifications such as phosphorylation and glycosylation are well established. Lipid modifications are less well characterized, however. It is estimated that as much as 0.5% of all cellular proteins may be prenylated1. Prenylation is the transfer of a 15-carbon farnesyl or a 20-carbon geranylgeranyl lipid chain to an acceptor protein containing the CAAX motif2. Prenylated proteins have been implicated in the progression of several human diseases including premature aging3, Alzheimer&#39;s4, cardiac dysfunction5, choroideremia6, and cancer7. The small GTPases, HRAS, NRAS, and KRAS1, nuclear laminins, and the kinetochores CENP-E and F are farnesylated proteins under the basal condition. Other small GTPases, namely RhoA, RhoC, Rac1, cdc-42, and RRAS are geranylgeranylated8, whereas RhoB can be farnesylated or geranylgeranylated9.
The small GTPase KRAS4b functions as a molecular switch, essentially transmitting extracellular growth factor signaling to intracellular signal transduction pathways that stimulate cell growth and proliferation, via multiple protein-protein interactions. There are two key aspects of KRAS4b biochemistry that are essential for its activity. First, the protein cycles between an inactive GDP and an active GTP bound state whereby it actively engages with effectors. Second, a C-terminal poly-lysine region and a farnesylated and carboxymethylated cysteine direct the protein to the plasma membrane, enabling recruitment and activation of downstream effectors. Mutant KRAS4b is an oncogenic driver in pancreatic, colorectal, and lung cancer10, and as such, therapeutic intervention would have a huge clinical benefit. Production of authentically modified recombinant protein that is farnesylated and carboxymethylated would enable biochemical screening using KRAS4b in combination with membrane surrogates such as liposomes or lipid nanodiscs11,12.
Farnesyl transferase (FNT) catalyzes the addition of farnesyl pyrophosphate to the C-terminal cysteine in the CAAX motif in KRAS4b. After prenylation, the protein is trafficked to the endoplasmic reticulum (ER) where the Ras converting enzyme (RCE1) cleaves the three C-terminal residues. The final step in processing is methylation of the new C-terminal farnesylcysteine residue by the ER membrane protein, isoprenylcysteine carboxyl methyltransferase (ICMT). Expression of recombinant KRAS4b in E. coli results in the production of an unmodified protein. Previous attempts to produce processed KRAS4b have been limited due to insufficient yields for structural or drug screening experiments or have failed to recapitulate the native full-length mature protein13,14. The protocol presented here utilizes an engineered baculovirus-based insect cell expression system and purification method that generates highly purified, fully processed KRAS4b at yields of 5 mg/L of cell culture.
Careful protein characterization is essential to validate the quality of recombinant proteins prior to embarking on structural biology or drug screening studies. Two key parameters of fully processed KRAS4b are validation of the correct prenyl modification and the availability of the farnesylated and carboxymethylated C-terminus (FMe) for interaction with membrane substitutes or lipids. Electrospray ionization mass spectrometry (ESI-MS) of the KRAS4b-FMe was used to measure the molecular weight and confirm the presence of the farnesyl and carboxymethyl modifications. Native mass spectrometry, where samples are sprayed with nondenaturing solvents, was used to demonstrate that KRAS4b-FMe was also bound to its GDP cofactor. Finally, surface plasmon resonance spectroscopy was used to measure the direct binding of KRAS4b-FMe with immobilized liposomes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.8 mL Safe-Lock Tubes, Natural</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>11 mm Cl SS Interlocked Insert Autosampler Vials</td>
			<td>Thermo Scientific</td>
			<td>30211SS-1232</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>AVANTI POLAR LIPIDS</td>
			<td>850457</td>
			<td>purchase as liquid stocks in chloroform</td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phospho-L-serine (POPS)</td>
			<td>AVANTI POLAR LIPIDS</td>
			<td>840034</td>
			<td>purchase as liquid stocks in chloroform</td>
		</tr>
		<tr>
			<td>5427R Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, HPLC Grade</td>
			<td>Fisher Chemical</td>
			<td>A998-1 1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>09689-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>Argon gas</td>
			<td>Airgas</td>
			<td>ARUP</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay Plate 384</td>
			<td>CORNING</td>
			<td>3544</td>
			<td></td>
		</tr>
		<tr>
			<td>Biacore T200 Instrument</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Snap-It Seals, T/S</td>
			<td>Thermo Scientific</td>
			<td>C4011-54B</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson Ultrasonic Bath</td>
			<td>Thermo Fisher</td>
			<td>15-336-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation Exchange Chromatography (CEX) column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29018183</td>
			<td>HiPrep SP Sepharose High Performance</td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma</td>
			<td>C3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Dyna Pro Plate Reader</td>
			<td>Wyatt Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Exactive Plus EMR Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>F0507-500Ml</td>
			<td>Use Reagent Grade or better</td>
		</tr>
		<tr>
			<td>Gilson vials 7x14 mm Tubes</td>
			<td>GE Healthcare</td>
			<td>BR-1002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass screw thread vials with PTFE foam liners</td>
			<td>Scientific Specialities</td>
			<td>B69302</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed/benchtop centrifuge</td>
			<td>Thermo Fischer Scientific</td>
			<td>05-112-114D</td>
			<td>capable of up to 4,000 xg</td>
		</tr>
		<tr>
			<td>His6-Tobacco Etch Virus (TEV) protease</td>
			<td>Addgene</td>
			<td>92414</td>
			<td>Purified as per Raran-Kurussi et al. (2017) Removal of Affinity Tags with TEV Protease. In: Burgess-Brown N. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY</td>
		</tr>
		<tr>
			<td>Immobilized Metal Affinity Chromatography (IMAC) column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28-9365-51</td>
			<td>HisPrep FF 16/10</td>
		</tr>
		<tr>
			<td>In-House Water Supply, Arium Advance</td>
			<td>Sartorius Stedim</td>
			<td></td>
			<td>Resistivity of 18 M&#937;0-cm</td>
		</tr>
		<tr>
			<td>Lipid extruder set with holder</td>
			<td>AVANTI POLAR LIPIDS</td>
			<td>610023</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Airgas</td>
			<td>NI-DEWAR</td>
			<td></td>
		</tr>
		<tr>
			<td>M110-EH microfluidizer</td>
			<td>Microfluidics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MabPac RP UHPLC Column, 4 um, 3.0 x 50 mm</td>
			<td>Thermo Scientific</td>
			<td>088645</td>
			<td></td>
		</tr>
		<tr>
			<td>MabPac SEC-1 Column, 5 um, 300 &#197;, 2.1 x 150 mm</td>
			<td>Thermo Scientific</td>
			<td>088790</td>
			<td></td>
		</tr>
		<tr>
			<td>MagTran software</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC Grade</td>
			<td>VWR Chemicals</td>
			<td>BDH20864.400</td>
			<td></td>
		</tr>
		<tr>
			<td>NGC Chromatography System</td>
			<td>BioRad</td>
			<td>78880002</td>
			<td>NGC QuestTM 100 Chromatography system</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail without EDTA or other chelators</td>
			<td>Millipore Sigma</td>
			<td>P8849</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber Caps type 3</td>
			<td>GE Healthcare</td>
			<td>BR-1005-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Series S Sensor Chip L1</td>
			<td>GE Healthcare</td>
			<td>29104993</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>13-400-519</td>
			<td>Absorbace at 280nm</td>
		</tr>
		<tr>
			<td>Ultra-15 Centrifugal Filter Units, 10K NMWL</td>
			<td>Millipore Sigma</td>
			<td>UFC901008</td>
			<td>PES membrane</td>
		</tr>
		<tr>
			<td>Ultracel 10K MWCO Ultra 0.5 mL Centrifuge Filters</td>
			<td>Amicon</td>
			<td>UFC501024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima - L80K</td>
			<td>capable of 100,000 xg</td>
		</tr>
		<tr>
			<td>Vanquish UHPLC (Pump, Column Hearter, and LC System)</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 2</td>
			<td>Fisher</td>
			<td>12-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, HPLC Grade</td>
			<td>Sigma-Aldrich</td>
			<td>270733-1L</td>
			<td>May use in-house water source (see below)</td>
		</tr>
		<tr>
			<td>Whatman GD/XP PES 0.45 mm syringe filter</td>
			<td>GE Healthcare - Whatman</td>
			<td>6994-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>Xcalibur QualBrowser</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>proteomics software</td>
		</tr>
	</tbody>
</table>

fully processed recombinant kras4b: isolating and characterizing the farnesylated and methylated protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

One of the largest variables in the protocol is the amount of expressed target protein (His6-MBP-tev-KRAS4b). This protocol was developed using an isolate from a Trichoplusia ni cell line, Tni-FNL17, adapted for suspension growth and weaned from serum. Given the wide range of results reported across the various insect cell lines with the baculovirus expression system, it is advisable that Tni-FNL be used, at least initially, to produced KRAS4b-FMe.
A dark prote...

Watch this Scientific Journal Video about Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein at JoVE.com

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Our protocol allows for isolation and separation of properly processed KRAS at high yield. The production of authentically farnesylated and carboxymethylated KRAS makes it possible to perform experiments that have KRAS in their membrane just like in mammalian cells. The high yield of the protocol sets it apart from previous work.We typically purify three to five milligrams of protein per liter of expression material. This allows us to conduct a variety of biophysical and structural biology experiments. KRAS is mutated in 20%of cancers and 90%in pancreatic cancers.So having a protocol to produce authentically processed protein is quite useful for developing drug screens so that you can study the actual protein as it would be on the membrane of cancer cells. This protocol is also very useful for studying other proteins that are farnesylated and fully processed in the cells. And as such, all you need to do is swap out the KRAS with the protein of interest and put it into the baculovirus.The critical steps are those around the cation exchange chromatography. Using the proper column and limiting the time the protein is in low salt buffer are essential for success. Understanding that the precipitation is not a snow globe type of event helps guide those new to the protocol.This is reinforced by showing the need to divide the cation exchange column load into multiple smaller loads. After lysing cells, clarifying the lysate, and purifying the target protein by IMAC, analyze the protein fractions using SDS page and Coomassie staining. Pool the peak fractions and dialyze the pool overnight against two liters of buffer D at four degrees Celsius.On the next day, remove the sample from dialysis and centrifuge it at 4, 000 times g for 10 minutes to remove any precipitate. The final dialyzed and clarified sample is often still hazy but can be applied to the CEX column without further processing. Prepare 20 milliliter cation exchange column by washing it with three column volumes of buffer G, then with three column volumes of buffer F.Next, dilute 20 milliliters of the dialyzed sample to a final sodium chloride concentration of 100 millimolar by adding 20 milliliters of buffer E and apply the sample to the exchange column.It is critical to dilute the small amount of the sample instead of all at once and the diluted samples should be applied to the column immediately after dilution to limit precipitation of the protein. Continue to load the column with freshly diluted sample as the previous dilution nears the end of the load. Then wash the column to baseline absorbance 280 with buffer F which typically require three column volumes.Elute the protein from the column with a 400 milliliter gradient from buffer F to 65%buffer G, collecting the eluent in six milliliter fractions. Once the gradient is complete, continue washing the column for an additional 1.5 column volumes of 65%buffer G.Choose the positive fractions based on both SDS page and Coomassie Blue stain analysis as well as inspection of the UV trace of the chromatogram. Then pool the protein and digest it with His6 TEV protease according to manuscript directions.After digestion and dialysis, load the protein onto a 20 milliliter IMAC column equilibrated with buffer A at three milliliters per minute. Collect seven milliliter fractions during this chromatography and wash the column with a total of three column volumes of buffer A or until baseline absorbance is reached. Elute the target protein with five column volume gradient of buffer C from 0%to 10%and collect seven milliliter fractions.Then identify the positive fractions with SDS page. When analyzed with SDS page, the clarified lysate should contain a dark band that migrates to about 65 kilodaltons which corresponds to the fusion protein His6-MBP-tev-KRAS4b. The co-expressed FNTAb migrates to 48 kilodaltons.The CEX step within the purification is critical because it reduces the extent of proteolysis and enriches the fully processed protein, but is the most complicated part of the protocol. A typical result from this step has a prominent peak three with the KRAS4b-FMe but elution profiles can be variable. Intact mass analysis using ESIMS confirm the precise molecular mass of the proteins and thereby the relative proportion of farnesylation or carboxymethylation.While typical final lots contained some detectable KRAS4b-FARN, this proportion was less than 15%in terms of peak height from this analysis. Native mass analysis was used to determine the mass of the KRAS4b bound to GDP. The sample solvent was exchanged for ammonium acetate to ensure a softer ionization allowing the native complex to stay intact.The interaction of KRAS4b-FMe and liposomes was measured with surface plasmon resonance to validate the farnesylation and carboxymethylation are required for KRAS4b-FMe to bind to membranes. As expected, KRAS4b-FMe bound to the liposomes while unprocessed KRAS4b did not. Minimizing the time the protein is exposed to low salt is the single most critical aspect of the protocol that affects the yield.The protein is only briefly at this low salt concentration following the dilution in buffer E.The protein can be used for a variety of biophysical experiments that use membrane mimetics like liposomes or nanodiscs to investigate which lipids KRAS interacts with. Using these data, we can start to extrapolate how KRAS interacts with the plasma membrane of the cell. Using purified KRAS-FMe, our colleagues were able to solve the x-ray crystal structure of KRAS in complex with a chaperone that is specific for the farnesylated and methylated form of this protein.

fully-processed-recombinant-kras4b-isolating-characterizing

Research

JoVE Journal

Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric&#160;&#946; subunit rings sandwiched between two heptameric&#160;&#945; subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome &#945; and &#946; subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete &#945;-ring and one complete &#946;-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

This protocol uses both subunit coexpression and postlysis subunit mixing for a more thorough examination of recombinant proteasome assembly.

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

A &quot;Dual-Addition&quot; Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...