Characterization and potential applications of a recombinant antifreeze protein from an antarctic yeast Glaciozyma antarctica produced in Pichia pastoris

Abstract

Ice recrystallization during thawing post-cryopreservation results in extensive cellular damage and ultimately leads to cell death and reduced cell viability. Antifreeze proteins (AFPs) are a group of proteins that allow organisms to survive in subzero environments. These proteins have thermal hysteresis and ice recrystallization inhibitory activities. In this present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Antarctic yeast, Glaciozyma antarctica, as a recrystallization inhibitor (RI) of ice growth and assessed its application as a cryopreservative of the fungal cutinase enzyme against freeze-thaw cycles. Recombinant Afp1 from G. antarctica, a psychrophilic yeast, has been produced in a methylotrophic yeast, Pichia pastoris, system that results in the expression of a hyper-glycoprotein (~55 kDa). Recombinant Afp1 exhibits antifreeze functions: thermal hysteresis (TH) and recrystallization inhibition where the highest TH values recorded for ~0.5°C at 10 mg/mL. The cryoprotective effects of Afp1 on purified recombinant cutinase showed that Afp1 can retain enzymatic activity up to ~20% when subjected to several cycles of freeze thawing. These findings indicate that Afp1 might act as a cryoprotective agent and thus, has great potential in biotechnology applications. © 2017, Malaysian Society of Applied Biology. All rights reserved.

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* To whom correspondence should be addressed.
Malays. Appl. Biol. (2017) 46(1): 213–218
CHARACTERIZATION AND POTENTIAL APPLICATIONS OF A
RECOMBINANT ANTIFREEZE PROTEIN FROM AN ANTARCTIC
YEAST Glaciozyma antarctica PRODUCED IN Pichia pastoris
MD TAB, M.1, HASHIM, N.H.F.1, ABU BAKAR, F.D.1, ILLIAS, R.2, NAJIMUDIN, N.3,
MAHADI, N.M.4 and MURAD, A.M.A.1*
1School of Biosciences and Biotechnology, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
2Department of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering,
Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
3School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
4Malaysia Genome Institute, Jalan Bangi Lama, 43000 Kajang, Selangor, Malaysia
*E-mail: munir@ukm.edu.my
Accepted 2 February 2017, Published online 31 March 2017
ABSTRACT
Ice recrystallization during thawing post-cryopreservation results in extensive cellular damage and ultimately leads to cell
death and reduced cell viability. Antifreeze proteins (AFPs) are a group of proteins that allow organisms to survive in sub-
zero environments. These proteins have thermal hysteresis and ice recrystallization inhibitory activities. In this present study,
we demonstrated the efficiency of a recombinant antifreeze protein from the Antarctic yeast, Glaciozyma antarctica, as a
recrystallization inhibitor (RI) of ice growth and assessed its application as a cryopreservative of the fungal cutinase enzyme
against freeze-thaw cycles. Recombinant Afp1 from G. antarctica, a psychrophilic yeast, has been produced in a methylotrophic
yeast, Pichia pastoris, system that results in the expression of a hyper-glycoprotein (~55 kDa). Recombinant Afp1 exhibits
antifreeze functions: thermal hysteresis (TH) and recrystallization inhibition where the highest TH values recorded for ~0.5°C
at 10 mg/mL. The cryoprotective effects of Afp1 on purified recombinant cutinase showed that Afp1 can retain enzymatic
activity up to ~20% when subjected to several cycles of freeze thawing. These findings indicate that Afp1 might act as a
cryoprotective agent and thus, has great potential in biotechnology applications.
Key words: antifreeze protein, Glaciozyma antarctica, recrystallization inhibition, cryoprotective
INTRODUCTION
Antifreeze proteins (AFPs) have evolved in cold-
adapted organisms to control ice crystal growth
upon exposure to sub-zero temperatures. It has been
suggested that the effect of these proteins results in
small sizes of ice crystals, which mitigates
mechanical damage to frozen tissues and cells that
can be caused by large ice crystals. These proteins
directly interact with ice surfaces and act to depress
the freezing point of body fluids, inhibit ice
recrystallization, or promote ice nucleation (Barrett,
2001). Several hypotheses have been advanced to
describe the mechanism of AFP binding to ice,
which occurs because of the diversity of structural
folds in this protein. Based on studies of insects and
fish AFPs, these include adsorption-inhibition,
hydrogen bond interactions and roles of
hydrophobic amino acids (Raymond & DeVries,
1977; Knight et al., 1993). The delicate control over
ice growth makes these proteins applicable to any
field that requires control of ice growth. AFPs from
fish (Types I–IV and antifreeze glycopeptide) have
been recognized as having the potential to be
widely used in most applications because they are
hypoallergenic (Kim et al., 2015). AFPs can be
applied in various foods and medical uses (Christner,
2010). They have been shown to improve the texture
of ice cream (Regand & Goff, 2006), increase the
quality and half-life of yeasts in frozen dough
(Zhang et al., 2007) and help to preserve meat
(Griffith & Ewart, 1995). AFPs have also been used
in cryosurgery and in blood and organ preservation
(Amir et al., 2003; Venkatesh & Dayananda, 2008).

214 ANTIFREEZE PROTEIN FROM AN ANTARCTIC YEAST Glaciozyma antarctica PRODUCED IN Pichia pastoris
Glaciozyma antarctica, a psychrophilic yeast
isolated from sea ice near Casey Research Station,
Antarctica, has been shown to produce AFP (Hashim
et al., 2013). The thermal hysteresis (TH) value
recorded from G. antarctica culture filtrate was
0.1°C and the Afp also exhibited recrystallization
inhibitory activity. Previously, recombinant
expression of G. antarctica Afp1 in Escherichia
coli was shown to result in the formation of
inactive inclusion bodies, which required further
manipulation to achieve biological activity.
Refolded recombinant Afp1 produced in E. coli
exhibited antifreeze activity that was lower than
that of the native form (TH=0.08°C) (Hashim et al.,
2013). However, because of poor yields obtained
from prokaryotic expression systems, a eukaryotic
expression system was chosen to overproduce this
protein. In this present study, we report on the
expression of G. antarctica Afp1 in methylotrophic
yeast, Pichia pastoris. This methylotrophic yeast
was used to express recombinant Afp1 because of
its ability to generate large amounts of properly
folded protein, ease of isolation and potential to be
produced as an extracellular protein (Cregg et al.,
1993). Additionally, the application of the
recombinant Afp1 recrystallization inhibition (RI)
activity as a cryoprotective agent against other
enzymes undergoing freeze-thawing was explored.
MATERIALS AND METHODS
Micro-organisms and plasmids
The methylotrophic yeast Pichia pastoris X-33
and expression vector pPICZαC used to express
Afp1 were purchased from Invitrogen (Carlsbad, CA,
USA). Plasmid pAFP1, harbouring a full-length
AFP1 sequence, was obtained from the Molecular
Mycology Laboratory, School of Biosciences and
Biotechnology, Faculty of Science and Technology,
UKM (Hashim et al., 2013).
Construction of pPICZαα
αα
αC_Afp1 and P. pastoris
transformation
The full sequence of AFP1 was amplified using
specific primers, AFP1_ClaI_F: 5'-TCA CCA TCG
ATG GCC ACC GCC ATC GA-3' and AFP1_XbaI_
R: 5'-GAA TTC ACT TCT AGA AAC CCA GGC
GCG-3', whereby the XbaI and ClaI restriction sites
were introduced at the 5'- and 3'-ends of the sequence
for cloning purposes. The 50 μL PCR reactions
contained the following contents: 2 μL pAFP1
harbouring full-length AFP1, 1× PCR buffer, 0.2 mM
dNTP, 20 pmol/μL forward and reverse primers,
1.5 mM MgCl2, and 0.5 U/μL Taq polymerase
(Invitrogen, Carlsbad, CA, USA). PCR was
conducted using the following thermocycling
conditions: initial denaturation 94°C for 5 min; 29
cycles of denaturation at 94°C for 45 sec, annealing
at 60°C for 30 sec and elongation at 72°C for 1 min
and a final extension at 72°C for 15 min. PCR
products were purified, cloned into linearized
pPICZαC and transformed into E. coli DH5α and
trans formants were then verified via PCR, restriction
enzyme analysis and sequencing.
To transform P. pastoris, a total of 5 μg
pPIC_Afp1 was linearized using PmeI, followed by
transformation into P. pastoris X-33 by electro-
poration with an Electroporator System 2510
(Eppendorf, AG, Hamburg, Germany). Transformants
were plated onto YPD medium that contained 100
μg/mL zeocin and was incubated at 30°C for 2
days. To assess correct integration, colonies were
screened by PCR using 5' and 3' AOX1 primers
according to the manufacturer’s instructions.
Positive transformants were plated onto YPD plates
that contained zeocin at different concentrations; 1
and 2 mg/mL were used to select multi-copy
integrants.
Expression of recombinant Afp1
In brief, recombinant P. pastoris strain X33 cells
that had been transformed by pPICZαC harbouring
the mature AFP1 gene were grown in Buffered
Minimal Glycerol Complex Medium (BMGY) that
contained (per litre) 10 g yeast extract, 20 g peptone,
1 M potassium phosphate (pH 6.0), 13.4 g yeast
nitrogen base, 400 μg biotin, and 10 mL glycerol.
Minimal medium (MM) consisted of (per litre) 10
mL 10×yeast nitrogen base, 10 mL 1× methanol and
0.2 mL 500× biotin as an expression medium;
expression was induced daily by the addition of
5 mL methanol at 28°C for 3 days. Culture
supernatants were confirmed by sodium dodecyl
sulphate-polyacrylamide (SDS) gel electrophoresis
(SDS-PAGE) and western blot analyses.
Protein purification
Crude protein was applied to an affinity
chromatography (Ni-NTA) column using 20 mM
Tris–HCl (pH 8), 150 mM NaCl and 10 mM
imidazole as binding buffer and with 20 mM Tris–
HCl (pH 8), 150 mM NaCl, and 500 mM imidazole
as elution buffer. Proteins were further purified by
size exclusion chromatography using a Superdex
S200 10/300 column (GE Healthcare, USA). All
purification steps were carried out using an AKTA
purifier (GE Healthcare, USA).
Antifreeze protein assay and cryoprotective effects
Recrystallization inhibition (RI) and the thermal
hysteresis assay (TH) using method described by
Kawahara et al (2007) were carried out using a
temperature-controlled freezing stage (Model THM
600, Linkham Scientific Instrument, UK) with a
temperature controller programming unit (Model

ANTIFREEZE PROTEIN FROM AN ANTARCTIC YEAST Glaciozyma antarctica PRODUCED IN Pichia pastoris 215
TMS 94, Linkham Scientific Instrument, UK). The
RI assay was carried out by the addition of 1 mg/
mL recombinant Afp1 to a mixture that contained
recombinant Glomerella cingulata cutinase (1 mg/
mL) (Wan Seman et al., 2014) to monitor the ability
of Afp1 to inhibit ice recrystallization. The
cryoprotective effects of Afp1 were further tested by
incubating Afp1 with recombinant protein at a ratio
of 1:1. Samples were maintained at -20°C for 3 h
and then were thawed at room temperature; the
experiment was repeated for five cycles. After five
cycles of freeze-thawing, samples were tested for
specific activity according to the protocol of Seman
et al (2014). Samples were then maintained at -20°C
and the freeze thaw cycles were then performed
every 12 h for two days.
RESULTS AND DISCUSSION
The cloning of a mature sequence of AFP1 and
transformation into P. pastoris was successfully
conducted. Figure 1 shows that recombinant Afp1
was expressed as a secreted protein. However,
recombinant Afp1 was produced at ~55 kDa, which
was larger than the predicted size of ~15 kDa.
Expression was confirmed by western blot analysis
using anti-His antibodies for which signals were
detected at ~55 kDa. This observation might
indicate this protein had been glycosylated. Our
previous findings also showed that native Afp1 from
G. antarctica was glycosylated when probed using
anti-Afp1 antibodies (Hashim et al., 2013).
The activity of purified recombinant Afp1 was
measured by observing changes in ice crystal
morphology and in the inhibition of ice
recrystallization. Generally, in the presence of
antifreeze proteins, ice crystal growth is inhibited,
resulting in an irregular ice crystal shape. This
occurs because the antifreeze protein can bind to
specific ice planes depending upon the comple-
mentary of its binding site with ice planes
(Kawahara, 2002). In contrast to samples without
antifreeze proteins, ice can expand when no
inhibitors are present in the solution, which results
in ice growth as round shaped crystals. Based on our
observations, the presence of recombinant Afp1-
changed ice morphology into a “flowery” shape
(Figure 2A) compared with solutions treated with
Proteinase K, in which round shape ice crystals
formed (Figure 2B & 2C).
Ice crystal formation was also modified by AFPs,
as the AFPs reduced the freezing point of a solution
without changing the melting point. This process
was defined as thermal hysteresis (TH) (Barrett,
2001). The TH value could be measured as the
difference between the temperature when single ice
crystals formed and the temperature for which the
ice started to change. The TH value recorded
for solutions that contained recombinant Afp1 was
0.5°C compared with solutions that contained
Proteinase K (0°C). The TH value was higher
Fig. 1. SDS-PAGE (A) and western blot analysis (B) of the expression of Afp1
in P. pastoris. 1: negative control (host Pichia pastoris); 2: Afp1 clone induce
with methanol; 3: Afp1 clone without induction. M: Prestained protein markers
(New England Biolabs, UK).

216 ANTIFREEZE PROTEIN FROM AN ANTARCTIC YEAST Glaciozyma antarctica PRODUCED IN Pichia pastoris
Fig. 2. Assays for ice crystal morphology and recrystallization inhibition of recombinant Afp1. The addition of proteinase K
inhibits Afp1 activity, which indicates ice crystal morphology. (A) Afp1 before treatment with proteinase K (B) Afp1 and
Proteinase K before incubation of (C) Afp1 after treatment with Proteinase K. (D) Recrystallization inhibition assay of cutinase
enzyme or cutinase enzyme with Afp1.
compared with recombinant rLeIBP from
Leucosporidium sp by 0.1°C (Lee et al., 2010),
and recombinant Afp1 produced in an E. coli
system (TH value=0.1°C) (Hashim et al., 2013). We
hypothesized that these observations might be a
consequence of hyperglycosylation activity on Afp1
that was produced in P. pastoris, which could not
be performed in a bacterial system. For Pseudomonas
putida AFP, the effects of its glycans towards AFPs
function has been observed when the activity was
reduced after glycans were removed from the protein
(Xu et al., 1998). To demonstrate that antifreeze
activity was associated with ice crystal morphology,
a series of experiments were carried out to determine
whether protease activity towards Afp1 affected the
morphology of the ice crystals. The ice crystals
grown in a solution that contained untreated Afp1
exhibited a hexagonal shape compared with Afp1
treated with Proteinase K, which had round shaped
ice crystals. The loss of antifreeze activity by
protease treatment indicated that the antifreeze
activity of Afp1 could disrupt ice crystal
morphology.
A RI activity assay was conducted to compare
the activity of ice grains in two different conditions:
a solution of cutinase enzyme without Afp1 and
another solution with a mixture of cutinase and
Afp1. Smaller ice grains were observed in solutions
that contained Afp1 after 1 h incubation at -6°C
compared with solutions that contained cutinase
enzyme alone without Afp1 (Figure 2D). This
observation established that Afp1 could inhibit ice
recrystallization. Ice recrystallization represents the
process of forming larger grains of ice upon
expansion from smaller ones. Since this process can
cause damage to cell membranes, the RI property
can increase the survival of cells after freeze-thaw
cycles (Raymond & Knight, 2003). These properties
appear to be favourable for the cold-adaptation
strategies of many polar organisms (Davies et al.,
2002; D’Amico et al., 2006). Microbial AFPs are
known to have superior activity for recrystallization
inhibition compared with other organisms because
microbes produce AFPs as part of their freezing
tolerance strategy to protect against ice injury
threats present at sub-zero temperatures (Lee et al.,
2010). Ice formation during preservation or cold
storage processes represents a major problem as it
causes nutrient loss, cell injury and denaturation
(Kim et al., 2015). We assessed the ability of Afp1
to function as a cryoprotective agent by incubating
recombinant G. cingulata cutinase with Afp1 and
subjecting it to several freeze–thaw cycles. Samples
were tested for its specific activity to assess whether
the activity was retained after a few cycles of freeze-
thawing. Cutinase in the presence of Afp1 retained
up to 20% of its activity after 2 days of incubation
compared with the control sample (no addition of

ANTIFREEZE PROTEIN FROM AN ANTARCTIC YEAST Glaciozyma antarctica PRODUCED IN Pichia pastoris 217
Afp1) (Figure 3). This revealed that Afp1 could
protect cutinase against protein degradation during
the freeze-thawing process. These observations also
suggest the potential of Afp1 to be applied as a
cryoprotective agent in industries such as food and
medicine. Currently, ongoing studies are aimed at
the production of large-scale recombinant Afp1 (i.e.,
in a bioreactor) and further assessments of its
potential applications as a cryoprotective agent.
CONCLUSION
In this study, recombinant Afp1 was successfully
produced via P. pastoris expression as glycoproteins
with a size of ~57 kDa. Further study showed that
recombinant Afp1 had the highest TH value at 0.5°C
and could modify the morphology of ice crystals.
Recombinant Afp1 lost its activity after treatment
with proteinase K. The cryoprotective effects of Afp1
on other enzymes showed that Afp1 could improve
enzyme stability after several rounds of freeze thaw
cycles.
ACKNOWLEDGEMENTS
This study was funded by the Ministry of Science,
Technology and Innovation (MOSTI) of Malaysia
under research grants 08-05-MGI-GMB001/1 and
07-05-MGI-GMB014.
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