Expression, Purification, and Antimicrobial Activity of S100A12

Summary

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Abstract

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Introduction

S100 proteins are a class of the EF-hand family of calcium binding proteins with a diverse array of functions¹. They are expressed in a tissue and cell specific manner, and regulate a broad spectrum of cellular functions^2,3. Unique to calcium binding proteins, S100 proteins exhibit both intracellular and extracellular functions^4,5. Within the cell, Ca²⁺ binding induces a conformational change that exposes a hydrophobic surface that specifically targets protein binding partners⁶. This intracellu....

Protocol

1. Expression of S100A12

1. Transform competent BL21 DE3 cells with a pGEMEX-S100a12 plasmid using a standard heat shock protocol¹⁸. Add 1 to 5 µL of plasmid to 50 µL of bacteria in a microcentrifuge tube on ice. Incubate for 20 min.
2. Heat shock the cells at 42 °C for 30 s.
3. Incubate the cells on ice for 2 min.
4. Add 500 µL of SOC media to the cells. Incubate at 37 °C with shaking at 250 rpm on an orbital shaker for 1 h.
5. Plate 150 µL of the transformation reaction on LB-agar medium (supplemented with 100 µg/mL ampicillin). Incubate for 12-16 h at 37 °C.
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Results

S100A12 expression and purification

A three-step purification produced ~40 mg of recombinant S100A12 from 50 mL of bacterial culture. The first step was an ammonium sulfate precipitation of endogenous E. coli proteins. This step was followed by anion-exchange chromatography (Figure1A). The protein is tracked by a SDS-PAGE stained with Coomassie Brilliant Blue (Figure 1B). .......

Discussion

An efficient protocol for both expression and purification of human S100A12 is presented. The E. coli expression system is the most common tool used for the production of recombinant proteins, particularly when mg quantities are required for biochemical and biophysical studies. A key enhancement of the procedure described here is the use of auto-induction media²⁶ which increases the yield of purified protein by a factor of almost thirty as compared to expression with standard Luria-broth .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
S100A12 Expression
Expression cells
BL 21 (DE3) competent cells	New England Biolabs	C25271
Autoinduction medium components
Yeast Extract	Research Products International	Y20020-250.0
NaCl	Research Products International	S23020-500.0
Tryptone	Research Products International	T60060-250.0
FeCl3	Sigma-Aldrich	F2877
MgSO4	Research Products International	M65240-100.0
Na2HPO4	Research Products International	S23100-500.0
KH2PO4	Research Products International	P41200-500.0
NH4Cl	Research Products International	A20424-500.0
Na2SO4	Research Products International	S25150-500.0
Glycerol	Research Products International	G22020-1.0
D-glucose	Research Products International	G32040-500.0
lactose	Sigma-Aldrich	L2643
Selection agent
ampicillin	Research Products International	A40040-5.0
S100A12 Purification
AKTA Start Chromatography System	GE	29-220-94
HiTrap Q Sepharaose Fast Flow	GE	17-5156-01
HiPrep 16/60 S-200 HR	GE	17-1166-01
Nanodrop lite spectrophotometer	Thermo Fisher Scientific	ND-LITE
Tris Base	Research Products International	T60040-1000.0
H. pylori Culture
Blood agar plates	Lab Supply Company	BBL221261
Brucella broth	Sigma-Aldrich	B3051
Cholesterol (250x)	Thermo Fisher Scientific	12531018
NaCl	Sigma-Aldrich	793566
CaCl2	Sigma-Aldrich	C4901
β-mercaptoethanol	Sigma-Aldrich	M6250
Tris Base	Sigma-Aldrich	T1503
Zinc Chloride	Sigma-Aldrich	229997