Team:Tokyo Tech/Antifreeze

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Revision as of 19:28, 21 October 2009

Achievement

• We achieved that AFP derived from mealworm was transformed into E.coli strains Origami 2 and was successfully expressed with its structure correctly folded. In addition, it had an activity in vitro analysis.

Anti-freeze protein
PDB code 1ezg

Introduction ~Why do we need Anti-freeze protein(AFP)~

The surface temperature of Mars is below freezing point almost all day. Most organisms will die if we simply send them to the present Mars. Therefore, we planed to produce Anti-freeze protein(AFP) into E.coli body. By producing AFP, bacteria will become surviving in low temperature. Then we will need less energy as a primary investment.

Material and Method

The characteristics of Anti-freeze protein(AFP)

Anti-freeze protein(AFP) is defined that a protein binds to single ice crystal specifically and inhibits its growth. When the temperature is below 0 ℃, water form 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 to lower the freezing point while havinng no effect on the melting point. This characteristics of AFP is called Thermal Hysteresis (TH), AFP is superior to other substances(for example NaCl), for it can greatly lower the freezing point. Then we thought that the organisms with AFP can lower the freezing point of their body fluid and is able to be less damaged.

We decided to use 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, which give specific TH activity about 10-100 times greater than that of fish AFP, and 300-500 times greater than those of other chemical compounds. We expressed this AFP in E.coli and examined its activity (and how to shift the mortality rate when bacteria bodies are in a repeated cycle of freeze thaw).

Anti-freeze protein
(PDB code 1ezg)

Experiments

• Designs of plasmids
  

We obtained the coding sequence of AFP which was chemically synthesized from GENEART. This gene digested with ClaⅠand 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 assembled on a Low Copy vector, pSB3K3. We constructed several parts which differ in only promoter sequence. We use placIq which is constitutive promoter, T7 promoter. Furthermore, inducible sequence LacO was added to T7 promoter.

• Expression and Purification

We introduced the AFP plasmids into the E.coli Origami 2 strain (Novagen). Single colonies were picked up from the transformed cells and inoculated into LB medium containing 30 μg/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 0.5 mM to over-express the EGFP-AFP fusion protein. Then cultures were shaken for a further 48 h 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 X mg/ml. We made solutions of five different concentration. 1ml of the aqueous solution was poured in a polymer test tube and layered 1ml of cilicon oil. Thermometer was attached to the outside the tube then the device was put into a thermostat bath. Temperature was slowly lowered (1.5 ℃/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 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 shown Fig 1. 1st and 2nd lanes are EGFP, 3rd and 4th lane are AFP-EGFP fusion protein. 1st and 3rd lanes with IPTG induction, and 2nd and 4th lanes were without IPTG induction. Molecular weight of EGFP-His is about 30 kDa, and AFP-EGFP fusion protein is about 40 kDa. The band of 3rd lane revealed around 40 kDa. So we regarded the protein as Antifreeze protein.

Fig 1.

• Assessing 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

References

• 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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