expression and purification of beetle antifreeze protein dafp-1 from
TRANSCRIPT
EXPRESSION AND PURIFICATION OF BEETLE ANTIFREEZE
PROTEIN DAFP-1 FROM BACTERIA Sandy Gordon*1, Jose Lopez2, Josh Fnu Arifin3, Alejandra Rios3, and Dr. Xin Wen3
1 East Los Angeles College, Monterey Park, CA 91754, 2 Pasadena City College, Pasadena, CA, 91106, 3 Dept. of Chemistry and Biochemistry, California State
University, Los Angeles, CA 90032
The beetle Dendroides canadensis produces several
isoforms of antifreeze proteins (DAFPs), that, when
combined together and/or with other enhancers, can create a
significant difference between melting and freezing points,
termed thermal hysteresis (TH). Though the effects of
DAFPs are well established, the exact mechanisms of how
these molecules work are still debatable. To achieve a better
understanding of these antifreeze proteins (AFPs), our
objective was to express DAFP-1, using Origami B (DE3)
strain of Escherichia coli (E. coli), and purify the protein
through several purification mechanisms.
INTRODUCTION
Antifreeze proteins (AFPs) depress the freezing point
of water in a non-colligative manner while leaving the
melting point unchanged; this difference between the
melting and freezing points is called thermal hysteresis
(TH) (Wang, 2005; Wang et al., 2009). This unique
ability of AFPs enable a wide variety of organisms such
as fish, plants, fungi and insects to be freeze avoidant or
freeze tolerant thus allowing them to survive in
otherwise deadly subzero climates. Given the
uncertainty of the exact mechanisms that take place, the
objective of this study was to express and purify
Dendroides canadensis’ antifreeze protein (DAFP),
specifically DAFP-1, to determine the crystal structure
and further analyze the function and thermal hysteresis
activity of DAFP-1. The possible applications of AFPs
range from extended tissue preservation of transplant
organs to increasing the freeze tolerance in crops (Wang,
2005).
METHODS
RESULTS
CONCLUSION
There are few theories explaining the
mechanism behind how antifreeze proteins
function; however, these are still debatable.
Furthermore, there is still much to be learned on
the enhancement of antifreeze protein activity.
For these reasons, it was necessary to express and
purify the beetle antifreeze protein DAFP-1 to
determine its crystal structure and further analyze
the thermal hysteresis activity.
FUTURE PLANS
We plan to continue studying and assisting in
the expression and purification of insect
antifreeze protein DAFP-1 with the purpose of
gaining a better understanding of its crystal
structure and on the enhancement of its thermal
hysteresis activity.
ACKNOWLEDGMENTS
We would like to thank Srikanya Jarugumilli for her feedback and
support . This project was supported by NIH GM086249 Grant and NIH
Bridges to the Future R25 GM 049001 grants.
Figure 1. Dendroides
Canadensis Figure 2. Model
tertiary structure of
DAFP-1 pictured with
arginine residues
Figure 3. 12% SDS-PAGE shows expression of DAFP-1. At 66
hours of incubation all 6 samples depict more intense bands at
25 kDa.
RESULTS
Figure 5. 12% SDS-PAGE shows His-tag from
DAFP-1 cleaved and before the his-tag was cleaved.
Figure 4. 12 % SDS-PAGE shows His-tag binding to uncut
protein as shown through first elution (lysis buffer B with 300
mM imidazole) , lane E1, with molecular weight of 25.0 kDa.
E2 E1 W F Post-C Pre-C M
#6 #5 #4 #3 #2 #1 M
25.0kDa
Purify using Ni-NTA SDS-PAGE
Cleave His-tag
Remove His-tag
through Ni-NTA
FPLC via gel-
filtration
Flow chart of experimental procedure used in protein expression
and purification. Ni-NTA, nickel-nitriloacetic acid column; SDS-PAGE,
sodium dodecyl sulfate polyacrylamide gel electrophoresis; His-tag,
polyhistidine-tag; FPLC; Fast protein liquid chromatography; MALDI-
TOF, Matrix-assisted laser desorption ionization-time of flight mass
spectrometry
Centrifuge and
collect pellet
Induce expression
of protein DAFP-1
Inoculate
colony
SDS-PAGE Grow bacteria cells
in LB media
containing
antibiotics
REFERENCES Wang, S., Amornwittawat, N., Juwita, V., Kao, Y., Dunman, J. G.,
Pascal, T. A., Goddard, W. A., Wen, X. (2009). Arginine, a key
residue for the enhancing ability of an antifreeze protein of the
beetle Dendroides canadensis. Biochemistry, 48, 9696-9703.
Wang, L. (2005). Identification and characterization of protein
enhancers of antifreeze proteins from overwintering beetle larvae
Dendroides Canadensis (Doctoral Dissertation). Retrieved from
collection of electronic master’s theses and doctoral dissertations
(ETD) available from the University of Notre Dame (etd-08302005-
141921 ).
Gofreed, Eric. Soldier Beetle- Dendroides canadensis (Image).
Available from: flickr. 18 Aug. 2011. Web. Accessed 13 Aug. 2012.
<http://www.flickr.com/photos/egofreed/6058780240/ >.
Figure 7. FPLC analysis of DAFP-1 zoomed curve. DAFP-1 was
separated from impurities through gel filtration chromatography.
mAU represents milli absorbance units. Reading taken at 280 nm
and flow rate was 0.5 ml/min.
maU
ABSTRACT
25.0 kDa
Pell Post Pre E2 E1 W F M 116.0 kDa 66.2 kDa 45.0 kDa 35.0 kDa
25.0 kDa
18.4 kDa
14.4 kDa
Figure 8. Matrix-assisted laser desorption ionization-time of
flight (MALDI-TOF) mass spectrum of DAFP-1.
Figure 6. FPLC analysis of DAFP-1 unfocused. DAFP-1 was
separated from impurities through gel filtration chromatography.
mAU represents milli absorbance units. Reading taken at 280 nm
and flow rate was 0.5 ml/min.
MALDI-TOF MS
8969.857
1689.841
4486.480
9193.806
9486.955