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

Summary

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

Abstract

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 'CAAX' 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.

Introduction

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's

Protocol

1. Protein purification

1. Prepare buffers A–H, as seen in Table 1.

Buffer solution	Buffering agent (all 20 mM)	pH	NaCl (mM)	imidazole (mM)	MgCl2	TCEP
A	HEPES	7.3	300	-	5	1

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.8 mL Safe-Lock Tubes, Natural	Eppendorf	22363204
11 mm Cl SS Interlocked Insert Autosampler Vials	Thermo Scientific	30211SS-1232
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)	AVANTI POLAR LIPIDS	850457	purchase as liquid stocks in chloroform
1-palmitoyl-2-oleoyl-glycero-3-phospho-L-serine (POPS)	AVANTI POLAR LIPIDS	840034	purchase as liquid stocks in chloroform
5427R Centrifuge	Eppendorf
Acetonitrile, HPLC Grade	Fisher Chemical	A998-1 1L
Ammonium Acetate	Sigma-Aldrich	09689-250g
Argon gas	Airgas	ARUP
Assay Plate 384	CORNING	3544
Biacore T200 Instrument	GE Healthcare
Blue Snap-It Seals, T/S	Thermo Scientific	C4011-54B
Branson Ultrasonic Bath	Thermo Fisher	15-336-1000
Cation Exchange Chromatography (CEX) column	GE Healthcare Life Sciences	29018183	HiPrep SP Sepharose High Performance
CHAPS	Sigma	C3023
Dyna Pro Plate Reader	Wyatt Technologies
Exactive Plus EMR Mass Spectrometer	Thermo Scientific
Formic Acid	Sigma-Aldrich	F0507-500Ml	Use Reagent Grade or better
Gilson vials 7x14 mm Tubes	GE Healthcare	BR-1002-12
Glass screw thread vials with PTFE foam liners	Scientific Specialities	B69302
High speed/benchtop centrifuge	Thermo Fischer Scientific	05-112-114D	capable of up to 4,000 xg
His6-Tobacco Etch Virus (TEV) protease	Addgene	92414	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
Immobilized Metal Affinity Chromatography (IMAC) column	GE Healthcare Life Sciences	28-9365-51	HisPrep FF 16/10
In-House Water Supply, Arium Advance	Sartorius Stedim		Resistivity of 18 MΩ0-cm
Lipid extruder set with holder	AVANTI POLAR LIPIDS	610023
Liquid nitrogen	Airgas	NI-DEWAR
M110-EH microfluidizer	Microfluidics
MabPac RP UHPLC Column, 4 um, 3.0 x 50 mm	Thermo Scientific	088645
MabPac SEC-1 Column, 5 um, 300 Å, 2.1 x 150 mm	Thermo Scientific	088790
MagTran software	Thermo Scientific
Methanol, HPLC Grade	VWR Chemicals	BDH20864.400
NGC Chromatography System	BioRad	78880002	NGC QuestTM 100 Chromatography system
Protease Inhibitor Cocktail without EDTA or other chelators	Millipore Sigma	P8849
Rubber Caps type 3	GE Healthcare	BR-1005-02
Series S Sensor Chip L1	GE Healthcare	29104993
Spectrophotometer	Thermo Fischer Scientific	13-400-519	Absorbace at 280nm
Ultra-15 Centrifugal Filter Units, 10K NMWL	Millipore Sigma	UFC901008	PES membrane
Ultracel 10K MWCO Ultra 0.5 mL Centrifuge Filters	Amicon	UFC501024
Ultracentrifuge	Beckman Coulter	Optima - L80K	capable of 100,000 xg
Vanquish UHPLC (Pump, Column Hearter, and LC System)	Thermo Scientific
Vortex Genie 2	Fisher	12-812
Water, HPLC Grade	Sigma-Aldrich	270733-1L	May use in-house water source (see below)
Whatman GD/XP PES 0.45 mm syringe filter	GE Healthcare - Whatman	6994-2504
Xcalibur QualBrowser	Thermo Scientific		proteomics software