expression and purification of beetle antifreeze protein dafp-1 from

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