Molecular cloning, expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

Noor Haza Fazlin Hashim, Suhaila Sulaiman, Farah Diba Abu Bakar, Rosli Md Illias, Hidehisa Kawahara, Nazalan Najimudin, Nor Muhammad Mahadi, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.

Original languageEnglish
Pages (from-to)1495-1505
Number of pages11
JournalPolar Biology
Volume37
Issue number10
DOIs
Publication statusPublished - 1 Aug 2014

Fingerprint

Antifreeze Proteins
Molecular Cloning
Antarctica
molecular cloning
Ice
ice
Yeasts
Inclusion Bodies
hysteresis
inclusion bodies
yeasts
Leucosporidium
Complementary DNA
Hot Temperature
fungal proteins
heat
Fungal Proteins
antifreeze proteins
hydrophobic bonding
Hydrophobic and Hydrophilic Interactions

Keywords

  • Heterologous protein expression
  • Homology modelling
  • Inhibition of recrystallisation
  • Thermal hysteresis

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)

Cite this

Molecular cloning, expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica. / Hashim, Noor Haza Fazlin; Sulaiman, Suhaila; Abu Bakar, Farah Diba; Illias, Rosli Md; Kawahara, Hidehisa; Najimudin, Nazalan; Mahadi, Nor Muhammad; Abd. Murad, Abdul Munir.

In: Polar Biology, Vol. 37, No. 10, 01.08.2014, p. 1495-1505.

Research output: Contribution to journalArticle

Hashim, Noor Haza Fazlin ; Sulaiman, Suhaila ; Abu Bakar, Farah Diba ; Illias, Rosli Md ; Kawahara, Hidehisa ; Najimudin, Nazalan ; Mahadi, Nor Muhammad ; Abd. Murad, Abdul Munir. / Molecular cloning, expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica. In: Polar Biology. 2014 ; Vol. 37, No. 10. pp. 1495-1505.
@article{9259c8f9e634425fbb6e6c6b3a5b8a8a,
title = "Molecular cloning, expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica",
abstract = "Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 {\%}). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.",
keywords = "Heterologous protein expression, Homology modelling, Inhibition of recrystallisation, Thermal hysteresis",
author = "Hashim, {Noor Haza Fazlin} and Suhaila Sulaiman and {Abu Bakar}, {Farah Diba} and Illias, {Rosli Md} and Hidehisa Kawahara and Nazalan Najimudin and Mahadi, {Nor Muhammad} and {Abd. Murad}, {Abdul Munir}",
year = "2014",
month = "8",
day = "1",
doi = "10.1007/s00300-014-1539-1",
language = "English",
volume = "37",
pages = "1495--1505",
journal = "Polar Biology",
issn = "0722-4060",
publisher = "Springer Verlag",
number = "10",

}

TY - JOUR

T1 - Molecular cloning, expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

AU - Hashim, Noor Haza Fazlin

AU - Sulaiman, Suhaila

AU - Abu Bakar, Farah Diba

AU - Illias, Rosli Md

AU - Kawahara, Hidehisa

AU - Najimudin, Nazalan

AU - Mahadi, Nor Muhammad

AU - Abd. Murad, Abdul Munir

PY - 2014/8/1

Y1 - 2014/8/1

N2 - Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.

AB - Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.

KW - Heterologous protein expression

KW - Homology modelling

KW - Inhibition of recrystallisation

KW - Thermal hysteresis

UR - http://www.scopus.com/inward/record.url?scp=84907883232&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84907883232&partnerID=8YFLogxK

U2 - 10.1007/s00300-014-1539-1

DO - 10.1007/s00300-014-1539-1

M3 - Article

VL - 37

SP - 1495

EP - 1505

JO - Polar Biology

JF - Polar Biology

SN - 0722-4060

IS - 10

ER -