Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Summary

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Abstract

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Introduction

The genus Burkholderia comprises more than 62 species, Gram negative organisms isolated from a wide range of niches, and it is divided in two main clusters^1,2. The first cluster includes human, animal and phytotrophic organisms, and most studies have focused on the pathogenic species of this group due to their clinical importance. The most pathogenic members are B. pseudomallei and B. mallei (which causes melioidosis and glanders respectively)^3,4 and opportunistic pathogens (the 17 defined species of the Burkholderia cepacia complex, BCC)⁵, which cause disease in cystic fibrosis (CF) and chr....

Protocol

1. Culture Growth (Days 1+2)

1. Grow a starter culture: 3 ml of Luria Bertani (LB) broth in a snap top 15 ml tube, at 37 °C rotator overnight. Dilute overnight growth to an OD₆₀₀ of 0.6. Add 1 ml of this dilution to 100 ml of LB broth in a 250 ml Erlenmeyer flask. Incubate at 37 °C with shaking at 250 rpm for 16 hr into stationary phase (SP), to better approximate, in batch culture, infection conditions and to assure that bacterial virulence factors under the control of stationary phase signal factors such as rpoS and quorum sensing, are expressed. Alternatively, bacteria grown at early SP or mid- or late-logarithmic phase can be used to ....

Results

Comparative analysis of the protein profiles extracted from the same bacterial culture on two different occasions showed similar patterns banding indicating successful protein extractions. Molecular weight proteins extracted ranged from 10-150 kDa. Figure 1 shows representative Coomassie blue staining gel of the whole-cell protein extractions from Burkholderia multivorans (a member of the BCC) clinical isolates grown in LB or Yeast/Manitol (YEM) broth and harvested from stationary phase.

Discussion

A method for proteins preparation has been described that can extract the majority of Burkholderia proteins with good reproducibility. This is demonstrated by obtaining the same protein profile from two independent preparations performed in different days using the same bacterial culture grown in LB or YEM broth as shown in Figure 1. Extraction was efficient for bacteria grown in liquid media; however we have not tested this method for bacteria grown on plates. This method was also used for furt.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Phenylmethanesulfonyl fluoride (PMSF)	Sigma	P7626	Toxic, corrosive
Acetone	Fisher	A18-1	Flammable
PBS Buffer	Bioscience	R028
Ethylenediaminetetraacetic acid (EDTA)	Fisher	BP118-500	toxic
Glass beads (0.1 mm)	BioSpec Products	11079101
Nalgene Oak Ridge Centrifuge tubes, polycarbonate (50 ml)	VWR	21009-342
MicroBCA protein extraction kit	Pierce	23235
Microcentrifuge Tubes 2.0 ml conical Screw cap Tubes with Cap and O-Ring	Fisher	02-681-375
2-D Clean-Up kit	GE Healthcare	80-6484-51
Urea	Invitrogen	15505-050	Irritant
CHAPS	Amersham Biosciences	17-1314-01
Dithiothreitol (DTT)	MPBiomedical, LCC	856126	Irritant
Immobiline DryStrips pH 4-7 24 cm	Amersham Biosciences	176002-46
Duracryl	Proteomic Solutions	80-0148	Very toxic, carcinogen
Ammonium persulfate	Fisher	BP179-100	Flammable, toxic, corrosive
N,N,N’,N’-tetramethylethylenediamine (TEMED)	Invitrogen	15524-010	Flammable
Sodium dodecyl sulfate (SDS)	Fisher	BP166-500	Acute toxicity, flammable
Agarose	Invitrogen	15510-027
Ethanol	Fisher	HC1100-1GL	Flammable, toxic
Acetic acid	Fisher	A491-212	Flammable, corrosive
Glutaraldehyde	Fisher	G151-1	Very toxic, corrosive, dangerous for the environment
Potassium tetrathionate	Sigma	P2926	Irritant
Sodium acetate	EM Science	7510
Silver nitrate	Sigma	209139	Corrosive, dangerous for the environment
Formaldehyde	Sigma	252549	Toxic
Sodium thiosulfate	Sigma	S7026
Tris Base	EMD	9230
Glycine	MPBiomedical, LCC	808831
Glycerol	MPBiomedical, LCC	800688
Methanol	Fisher	A412-4	Flammable, toxic, health hazard
Sodium carbonate anhydrous	EMD	SX0400-3	Toxic
Mineral oil	ACROS	415080010
Equipment
JA-20 ultracentrifuge rotor	Beckman Coulter	334831
Mini Beadbeater	Biospec Products	3110BX
Ettan IPGphor II Isoelectric Focusing System and accessories	GE Healthcare	80-6505-03	www.amershambiosciences.com
Ettan DALT Large Vertical electrophoresis system	GE Healthcare	80-6485-27	www.amershambiosciences.com