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

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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><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&amp;Omega;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 &amp;Aring;, 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

Research

JoVE Journal

Biochemistry

5.6K Views.  Frederick National Laboratory for Cancer Research. Ons protocol maakt isolatie en scheiding van goed verwerkte KRAS bij hoge opbrengst mogelijk. De productie van authentiek vernesylated en carboxymethylated KRAS maakt het mogelijk om experimenten uit te voeren die KRAS in hun membraan hebben, net als in zoogdiercellen. De hoge opbrengst van het protocol onderscheidt het van eerder werk.We zuiveren meestal drie tot vijf milligram eiwit per liter expressiemateriaal. Dit stelt ons in staat om een verscheidenheid van biofysische en structu...

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

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Genetics

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Cancer Research

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Immunology and Infection

Isolation of Stably Transfected Melanoma Cell Clones

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated cell transfection as it can lead to a heterogeneous cell population and variable transfection efficiency, affecting reproducibility. Moreover, they are preferable for large-scale analyses. The generation of stable cell clones is useful for a wide range of applications, such as research on gene functions and recombinant protein production. There are a few methods to obtain a homogenous cell population upon initial transient transfection. Here, we describe the isolation of single cell clones with glass cylinders. Although this method has been known for some time, there are a few crucial steps, and neglecting them may lead to failure. We have successfully used this method to obtain clones stably overexpressing a protein of interest (POI) or with knockout of a gene of interest (GOI). We describe preparation steps such as the optimization of selecting drug concentrations, preparation of glass cylinders, and validation of whether the obtained clones have the desired change in the expression of the GOI by PCR, western blot analysis, immunostaining, or gDNA sequencing (depending on the type of derived clones). We also discuss the phenotypic heterogeneity of well-established cell lines as this might be an issue in obtaining stable cell clones.

A protocol is described for isolating stably transfected melanoma cell clones using glass cylinders. We focus on practical advice that may be decisive for the success of the entire procedure.

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

Biology

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

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

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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

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