Team:Tokyo Tech/Antifreeze

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Revision as of 13:09, 19 October 2009

Introduction of AFP

Why fish living in the Arctic Ocean don’t die even the temperature of the sea is below freezing point? Most of the life cannot live in the climate which temperature is below the freezing point because water in their body freeze and these freezing damages harm their bodies. Everyone may have thought about this question once. Then, how the fish survive in such a severe climate? The clue of this question is a protein discovered in 1969. It is called Antifreeze Protein. It was firstly discovered in a fish living in the Antarctic Ocean, and until today, AFP is discovered from many other creatures and studied much more deeply. In this page, we will introduce AFP’s characteristics.

The characteristics of AFP

AFP is defined that a substance binds to single ice crystal specifically and inhibit its growth. When the temperature is below 0℃, water become ice nucleation. Then it grows up to bigger ice block. In the presence of AFP, on the other hand, AFP binds the ice nucleation very specifically and inhibits growing. For that reason AFP is thought that it lowers the freezing point while leaving the melting point unchanged. These characteristics of AFP is called Thermal Hysteresis (TH), AFP is superior to other substances, for it can greatly lower the freezing point. So we thought that the creature which enables to produce AFP (in their body) can lower the freezing point of their body fluid and is able to be less damaged so that it will survive.

Why we noticed AFP?

The surface of the Mars is below freezing point almost all day. Most of the life will die if we simply send them to the present Mars. So we planed to transform AFP to E.coli body. If it can produce AFP, coli will survive in low temperature and we will use less energy to primary investment. We focused on the AFP of the yellow mealworm Teneblio molitor. This AFP consists of 84 amino acid, right handed β-helix with 12 residues per coil. This protein has many intramolecular disulfide bonds and a rigid array of threonine side chains, giving it specific TH activity about 10-100 times greater than that of fish AFP, 300-500 times greater than other chemical compounds. We expressed this AFP in E.coli and examined its activity (and how to shift the mortality rate when fungus bodies are in a repeated cycle of freeze thaw).

Experiments

Designs of vectors We assembled the cording sequence of AFP synthetically from GENEART. This gene digested with ClaⅠ, then it was integrated in the downstream of promoter, RBS, and eGFP for fusion protein. In addition, we put a His tag for purification at the end of the insert. This series of parts is on a Low Copy vector, pSB3K3. We constructed several parts which differ in only promoter sequence. We used constitutive PlacIq, constitutive in the presence of T7 RNA polymerase T7 promoter. Furthermore, inducible sequence LacO was added to T7 promoter.

Expression and Purification

We transformed the AFP vectors into the E.coli Origami2 strain (Novagen). Single colonies were picked up from the freshly transformed cells and inoculated into LB medium containing 1.2μg/ml kanamycin. Cultures were grown at 37℃ with shaking (about 180rpm) to OD=0.5. The temperature was lowered to 15℃ and cultures were shaken for 1h. IPTG was added to a final concentration of 0.5mM to over-express the eGFP-AFP fusion protein. Then cultures were shaken for a further 48h at 15℃ and harvested by centrifugation. These fungus bodies were divided and some were checked for protein expression by SDS-PAGE and others were purified. The x g of purified protein was dissolved in Yml of Milli-Q and checked its freezing point.

Measure of the freezing point

The purified AFP was dissolved into Milli-Q to Xmg/ml. 1ml of the aqueous solution was poured in a polymer test tube and layered 1ml of oil. Thermometer was attached to the outside the tube then the device was put into a thermostat bath. Temperature was slowly lowered (1℃/min) and we recorded that of inner solution. The series of this operation was tried about 30 times. We calculated the average of the freezing point, and defined as (E.coli produced) AFP’s freezing point.

Results

Expression of antifreeze protein The fusion proteins were (readily) detected by SDS–PAGE stained with CBB (Coomassie Brilliant Blue). The result is below. 1st and 2nd lanes are eGFP, 3rd and 4th lane are AFP-eGFP fusion protein, the last lane is PET. 1st and 3rd lanes were inducted IPTG, and 2nd and 4th lanes were without IPTG induction. We already know from sequence EGFP-His is x kDa, and AFP-EGFP fusion protein is x kDa. The band of 3rd lane revealed around x kDa. So we regarded the protein as Antifreeze protein.

Assessment the activity of antifreeze protein

We measured TH of a solution of anti-freeze protein purified from Origami 2. Calorimetric measurements of anti-freeze protein in Milli-Q water revealed that the protein depresses the freezing point. Supercooled water gave a sharp freezing endotherm with an onset of x °C, whereas water with TmTHP showed an endotherm with an onset of x °C. So the anti-freeze protein has an activity of TH.

Discussion

Reference

M. Bar, R. Bar-Ziv, T. Scherf, D. Fass, Efficient production of a folded and functional, highly disulfide-bonded β-helix antifreeze protein in bacteria, Protein Expression & Purification, 2006 C. Yue, Y. Zhang, Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli, Molecular Biology Rep (2009) 36:529-536

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