Enriching Subcellular Proteins in Leptospira Using a Triton X-114-Based Fractionation Approach

Summary

Though prokaryotes lack an organellar system, there are subcellular regions with localized proteins. Quantitative and qualitative analysis of the enzymes and proteins in the subcellular regions is important for developing drug and vaccine targets. Here, we illustrate the subcellular fractionation of Leptospiral proteins using Triton X-114 and its analysis.

Abstract

Prokaryotes lack precise subcellular organelles but exhibit distinct regions such as cytoplasm, inner membrane, periplasm, and outer membrane, where most biochemical and physiological functions are organized. Thus, understanding the functional characteristics of proteins necessitates elucidating their subcellular localization. However, extracting subcellular proteins from gram-negative bacteria poses challenges due to their complex phospholipid bilayer. Although Triton X-114 has shown promise in outer membrane protein (OMP) extraction, a concise protocol remains elusive. This protocol demonstrates a step-by-step workflow for extracting subcellular proteins using the spirochete Leptospira as a model. This technique, featuring subcellular fractionation and phase separation, yields distinct fractions for cytoplasmic, outer, and inner membrane proteins. The detergent Triton X-114 is well-suited for phase separation due to its optimal cloud point temperature (~22 °C) and low critical micelle concentration (CMC), enabling efficient extraction and purification of native proteins with minimal denaturation. Notably, the analysis reveals the efficiency in discriminating proteins from the inner and outer membranes distinct from the cytoplasm.

Introduction

Subcellular proteomic (SCP) analyses play a pivotal role in elucidating bacterial pathogenesis by analyzing the proteome within specific subcellular compartments of bacterial cells. This subcellular proteomics approach characterizes bacterial proteins within distinct compartments, reducing complexity by excluding highly abundant proteins and providing insights into their organization and proportional abundance^1,2. For instance, employing subcellular fractionation and LC-MS/MS techniques, a study on Shewanella oneidensis unveiled a substantial portion of its proteome, furnishing crucial details regardi....

Protocol

NOTE: This protocol utilizes Leptospira interrogans for the subcellular protein extraction experiment. While this protocol can be adapted for use with other gram-negative bacteria, optimization may be necessary depending on the specific research objectives. Table of Materials and Table 1 provide the list of chemicals and equipment, along with the compositions of buffers and reagent solutions, respectively, while Table 2 outlines laboratory facilities.

1. Leptospira strain and culture conditions

1. Obtain the L. interrogans serogroup Icter....

Results

In this experiment, we employed the spirochete Leptospira as a model organism to extract subcellular compartments. We employed a strategic methodology anchored in cloud point and critical mycelial concentration (CMC), harnessing the effectiveness of Triton X-114, a non-ionic detergent (Figure 1). Before proceeding further, protein estimation was conducted to assess the concentration in each fraction for a single biological replicate. We observed a concentration ratio of 3:1:1.3 in A.......

Discussion

Subcellular fractionation techniques play a pivotal role in the isolation of discrete cellular constituents for subsequent analysis. Triton X-114, a non-ionic detergent, is commonly used in these techniques because of its ability to partition samples into distinct phases, allowing for the separation of various cellular components³². Triton X-114 has been particularly useful in extracting membrane proteins (in native form) from a variety of sources, including urine extracellular vesicles, Golgi mem.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Sigma	695092
Acetone	Sigma	32201
Acrylamide	Sigma	A-3553
Ammonium persulfate	Sigma	A-3678
Balance	SHIMADUZ	Z741079
Bis Acrylamide	Sigma	M7279
Bovine serum albumin fraction V (BSA)	Sigma	A-8022
Bromophenol Blue	Sigma	B-8026
Centrifuge Tube (50 mL)	Tarsons	500020
Conical Flask with Cap (150 mL)	Tarsons	431030
Cooling centrifuge	Eppendorf	5355316200
Coomassie brilliant blue	Sigma	1.12553
Dark field microscope	ZEISS	GmbH37081
Delicate Task Wipes	Kimtech Science	34155
Dry bath	Eppendorf	535531620
Ellinghausen–McCullough–Johnson–Harris (EMJH) medium, Difco	Difco	279410
Gel trays: Gel trays must be at least 2cm wider in dimensions and have smooth and flat surface with lid.	Hoefer	Not applicable
Glass Beake (150 mL)	Tarsons	421030
Glycine G8790	Sigma	G-8898
Immobilon Blotting Filter Paper	Millipore	IBFP0813C
Immunoblot semi-dry setup	ATTA	AE-6675
Incubator - For incubating the gel trays for 18 hours at 37°C.	EYELA	SLI-700
Laminar air flow	Not applicable	Not applicable
Luminol	Sigma	09253
Magnesium chloride M8266	Sigma	M8266
Magnetic stirrer	TARSONS	6040
Methanol	Sigma	17995-7
Micropipette Tips (10 µL–10 mL)	Axygen	T-10, T-200-C,T-1000-C, T-10ML-C
Micropipettes	Eppendorf	EP2231300010
Microtubes (1.5 mL Safe-Lock)	Eppendorf	EP022363212
Mini spin	Genaxy	GEN-MINI-6K
Multimode reader	BMG LABTECH	S/N 413-3877
Nonfat-Dried Milk, Bovine	Sigma-Aldrich	M7409
P-Coumaric acid	Sigma	C9008
Phosphate buffered saline	Sigma	P4417
Refrigerator	LG	GC-B217BLJ2
Rocker - Rocking or rotary shaker with low speed for staining the gel.	TARSONS	4080
SDS-PAGE SETUP- Electrophoresis Apparatus	Hoefer, Inc.	SE300-10A-1.0	Hoefer model ‘miniVE’ which can cast 10 cm x 8 cm sized gel of thickness 1 mm.
Sodium chloride	Sigma	5886
Sodium dodecyl sulfate (SDS)	Sigma	L-4396
TEMED	Sigma	T-9281
Triton X-114	Sigma	648468
Trizma Base (TRIS) T6066	Sigma	T6066
Vortex	TARSONS	3020