Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

Summary

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

Abstract

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Introduction

The quaternary structure of a protein plays a crucial role in many cellular processes. Signaling pathways, gene expression, and enzyme activation/deactivation all rely on the proper assembly of protein complexes ^1-4. This process also known as homo- or hetero-oligomerization is due to irreversible covalent or reversible electrostatic and hydrophobic protein-protein interactions. Oligomerization not only diversifies the different cellular processes without increasing the genome size, but also provides a strategy for proteins to build stable complexes that are more resistant towards denaturation and degradation ⁵. Defects in oligomerization have an....

Protocol

NOTE: This protocol is based on the previously published non-denaturing PAGE protocol ¹². In that study, the oligomeric state of the MxA protein was assessed using either Vero cells overexpressing MxA or IFN-α-stimulated A549 cells expressing endogenous MxA. The protocol described below can be used to analyze the oligomeric state of any protein in addition to MxA. However, further optimization may be required.

1. Preparation of Cell Lysate for Non-denaturing PAGE

NOTE: To analyze the oligomeric state of the human MxA protein in either Vero or A549 cells, 1.0 x 10⁶ cells were harvested. De....

Results

Using non-denaturing PAGE, we analyzed the oligomeric state of the human wild type MxA, the dimeric interface mutants MxA(R640A) and MxA(L617D) as well as the monomeric interface mutant MxA(M527D) from cell lysates ¹². Cells were lysed in a buffer containing 1% octylphenoxypolyethoxyethanol (NP-40) and iodoacetamide to ensure protein solubilization and protection of free thiol groups (see Figure 1). As described before, salt and small metabolites were removed by dialysis ¹⁹. Protein.......

Discussion

Here we describe a simple method that allows the rapid determination of the oligomeric state of proteins expressed in mammalian cells by non-denaturing PAGE followed by Western blot analysis. The major advantage of this approach is that the oligomeric state of a given protein can be determined from whole cell lysates without prior protein purification. This may be important for proteins that oligomerize or exert their function in association with auxiliary factors. In addition, the proteins are still in their native stat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Slide-A-Lyzer MINI Dialysis Units, 10K MWCO, 0.5 ml	Thermo Fisher Scientific	69570	Pre-equilibrate in dialysis buffer (if Glycerol removal is desired) Can be self-made according to Fiala et al. 2011
4–15% Mini-PROTEAN TGX Precast Protein Gels, 10-well,	Bio-Rad	456-1083	Pre-run in running buffer to adjust buffer system
cOmplete, Mini, EDTA-free	Roche	11836170001	use 1 tablet per 50 ml
PBS, pH 7.4  bottle a 500 ml Gibco	Thermo Fisher Scientific	14190-094
Ponceau S solution	Sigma-Aldrich	P7170	TOXIC wear gloves and protect eyes
NativeMark Unstained Protein Standard  50 µl	Invitrogen	P/N 57030	load 5 µl/well
A549 cells	ATCC	ATCC CCL185	Grow in growth medium (see Table 1)
Vero cells	ATCC	ATCC CCL81	Grow in growth medium (see Table 1)
anti-Mx1 antibody	Novus Biologicals	H00004599_D01P	Use at a 1:1,000 dilution
ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from donkey)	GE-Healthcare	NA934V	Use at a 1:10,000 dilution
0.5% Trypsin-EDTA (1x)        Life Technologies	Thermo Fisher	15400-054
Iodoacetamide   5 g	Sigma-Aldrich	I-6125	stock  100 mM
Bromphenolblue	Sigma-Aldrich	B0126-25G
DMEM +4.5g/l Gluc,+L-Glut,+Pyruvate life technologies	Thermo Fisher Scientific	41966-029
Pen  Strep 100 x     100ml               life technologies	Thermo Fisher Scientific	15140 - 130
Glutamax 100x Stock, 100 ml     life technologies	Thermo Fisher Scientific	350500-038
Fetal Bovine Serum, Dialyzed , US Origin 500 ml Gibco Lot:42G9552K	Thermo Fisher Scientific	10270-106
Cellulose filter paper	Bio-Rad	1703965
PVDF blotting  membrane	GE-Healthcare	10600022
Tris(hydroxymethyl)aminomethane	Biosolve	0020092391BS
sodium fluoride (NaF)	Sigma Aldrich	S-7920
NP-40	Calbiochem	492015
cOmplete, Mini, EDTA-free	Roche	11836170001
Tween 20	Calbiochem	6555204
CHAPS 10% solution	Amresco	N907
DL-Dithiothreitol (DTT)	Sigma Aldrich	43819
Glycine	Biosolve	0007132391BS
sodium orthovanadate (Na3VO4)	Sigma Aldrich	450243
Glycerol	Sigma Aldrich	G7757
β-Glycerophospate	Sigma Aldrich	G9422
Milk powder	Migros/Switzerland
Methanol	Millipore	1.06009
sodium cloride (NaCl)	Sigma Aldrich	71380
magnesium chloride (MgCl2)	Amresco	288
Sodium dodecyl sulphate (SDS)	Sigma Aldrich	L4509
sodium hydroxide (NaOH)	Sigma Aldrich	S-8045