Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Summary

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Abstract

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

Introduction

Ice-binding proteins (IBPs) are a diverse family of protective proteins that have been discovered in a number of organisms including plants¹, insects², fish³, and microbes⁴. The key feature of these proteins is their unique ability to specifically and efficiently adsorb to ice crystals, modifying their growth. IBPs have several documented properties, with the two most well characterized being thermal hysteresis (TH) and ice-recrystallization inhibition (IRI). TH activity is more readily observed in IBPs produced in freeze-intolerant animals. This activity results in lowerin....

Protocol

1. Splat Apparatus Setup

1. Fill a temperature-programmable circulating water bath with ethylene glycol (50% v/v in water).
   NOTE: Green automotive ethylene glycol can be used.
2. To assemble the external chamber, use insulated plastic tubing to attach the water bath to a double-walled glass bowl. Insulate the glass bowl with polystyrene foam and cover with a plastic petri dish lid. Cut a 3-4 inch hole in the bottom of the polystyrene chamber so that the light source can be seen through the glass chamber.
3. Mount the external chamber on a dissection microscope, fitted with a camera port and a 4X polarized objective lens with a 10x ocular ....

Results

For ease of set-up, Figure 1 and Figure 2 are included as visual representations of the equipment used for IRI analysis and ice-affinity purification, respectively. Results of IRI analysis using extracts collected from mustard weed with and without IBPs are depicted in Figure 3. These results show that extracts collected from mustard weed, which is not freeze-tolerant, were unable to.......

Discussion

For the successful analysis and purification of IBPs, it is important to understand the temperature-sensitive nature of some of these proteins. Certain plant IBPs become unstable at temperatures above 0 °C, resulting in unfolding, precipitation and inactivity. In order to obtain active IBPs, it is often critical that plants are processed in a cold room (~4 °C) and samples are kept on ice during experimentation. Another factor to consider when using whole-cell crude lysates is the degradation of proteins by endo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifugetubes	Fisher	05-408-129
Adjustable lab jack	Fisher	S63080
Benchtop centrifuge	Desaga MC2
Brass probe	Custom built
Camera/camera port	Canon		Canon Power Shot SX110 digitial camera; custom built microscope port
Cheesecloth	Purewipe/Fisher Scientific	06-665-25A
Concentration tubes (0.5 mL)	EMD Millipore	UFC501008
Concentration tubes (15 mL)	EMD Millipore	UFC900308
Conical tubes (50 mL)	Thermo Fisher	AM12502
Cooling block	VWR	13259	Use a metal heating block
Dehumidifier	Whirlpool		50 pint Energy Star dehumidifier; purchase from local supplier
Dessciation beads	t.h.e. Dessicant/VWR	EM-DX0017-2	6-8 mesh size; 100% indicating
Dissection microscope	Olympus Tokyo
Double walled glass bowl	Generic		Purchase from local lab glassware supplier
Dry ice	Generic		Use local supplier; hazardous
EDTA-protease inhibitor tablets	Sigma Aldrich	11836170001	Roche cOmplete mini
Ethylene glycol	Generic		Green automotive ethylene glycol can be purchased from any local hardware store (i.e. Home Depot)
Hexane	Sigma Aldrich	296090	Anhydrous, 95%; hazardous
Immersion oil	Sigma Aldrich	56822
JA10/20 centrifuge	Beckman
Large plastic Petri dish	Generic
Liquid nitrogen	Generic		Use local supplier; hazardous
Magnetic stir plate	Hanna Instruments	HI190M
Microscope cover slides	Fisher	12-542A
Plastic tube	Generic		Purchase PVC pipe from local hardware store
Polarized film	Edmund Optics	43-781
Polystyrene foam	Generic		Can be constructed from polystyrene shipping boxes
Poreclain mortar and pestle	Fisher	FB961
PVPP	Sigma Aldrich	77627	110 µm particle size
Retort Stand	Fisher	12-000
Small stir bar	Fisher	14-513-51
Temperature-programmable water bath	VWR	13271-118
Vacuum grease	Dow Corning/Sigma Aldrich	Z273554
Vinyl tubing	Generic