Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

Summary

This protocol focuses on the use of bacterial spores as a "live" nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

Abstract

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

Introduction

Display systems are aimed at presenting biologically active molecules on the surface of microorganisms and finding applications in a variety of fields, from industrial to medical and environmental biotechnologies. In addition to phages^1,2 and cells of various Gram-negative and -positive species^3,4,5,6,7, bacterial spores have also been proposed as display systems by two approaches^8,9.

Protocol

1. Adsorption reaction

1. Incubate 2, 5, and 10 µg of mRFP with 2 x 10⁹ of B. subtilis wild-type purified spores in 200 µL of binding buffer, 50 mM sodium citrate, pH 4.0 (16.7 mM sodium citrate dihydrate; 33.3 mM citric acid) for 1 h at 25 °C on a rocking shaker (Figure 1).
2. Centrifuge the binding mixtures (13,000 x g for 10 min) to fractionate pellets (P2, P5, and P10) and supernatants (S2, S5, and S10). Store the supernatants for the indirect evaluation of the efficiency of adsorption described in step 3.2.
3. Wash the pellets 2x with 200 µL of binding buffer and....

Results

Successful adsorption can be assessed by western blotting. Upon the reaction, the mixture is fractionated by centrifugation and washed, and the pellet fraction (Figure 1) is used to extract the surface proteins. The extract is fractionated by SDS polyacrylamide gel electrophoresis (PAGE), electrotransferred to a polyvinylidene fluoride (PVDF) membrane, and reacted against primary and secondary antibodies. The presence of proteins of the expected size, only in.......

Discussion

This spore adsorption protocol is very simple and straightforward. The reaction is strictly dependent on the pH of the reaction buffer and the efficiency of adsorption is optimal at acidic pH values (pH 5.0 or lower). At neutral pH conditions, the efficiency of adsorption is low, and at alkaline pH values, adsorption may not occur. Optimal adsorption is obtained using a volume of 200 µL in 1.5 mL tubes (or keeping a similar ratio) on a rocking shaker.

Adsorption is very tight, and washes .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1% Poly-L-lysine solution	Sigma	P8920
0.45 µm Nitrocellulose Blotting Membrane	Sartorius	M_Blotting_Membranes
100× objective UPlanF1	Olympus	microscope equipment
Bacillus subtilis strain NCIB3610	Bacillus Genetic Stock Center	3A1
BD ACCURI C6 PLUS	BD	flow cytometer
BX51	Olympus	Fluorescent microscope
Clarity	Biorad	1705060
DP70 digital camera a	Olympus	microscope equipment
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC	Thermo fisher	F-2754
Goat Anti-Rabbit IgG H&L (HRP)	Abcam	ab6721
Monoclonal Anti-polyHistidine−Peroxidase antibody	Sigma	A7058-1VL	used to detect adsorbed proteins presenting a 6xhistidine-tag at C- or N- terminal
SecureSlip glass coverslip	Sigma	S1815-1PAK
Superwhite Uncharged Microscope Slides	VWR	75836-190
U-CA Magnification Changer	Olympus	microscope equipment