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

<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>

The authors have nothing to disclose.

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 ...

fully-processed-recombinant-kras4b-isolating-characterizing

Universidad de Cartagena UDEC

Research

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Biochemistry

5.6K Views.  Frederick National Laboratory for Cancer Research. 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

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

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 ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

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 ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

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 ...

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

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Chapters in this video

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

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

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

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

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

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

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy