Difference between revisions of "Team:Queens Canada/AFP Scaffold"

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Revision as of 23:38, 15 September 2015

THE ICE QUEEN

Among the challenges in preserving harvested organs for transplantation is the time limit for which tissues remain undamaged outside of the human body. A potential solution to this limitation is the introduction of phenomena used by other organisms to enable sub-cooling of cells. In 2005, researchers in Israel and Californa1,2 successfully preserved rat hearts in University of Wisconsin (UW) solution3 with unmodified Type I and Type III antifreeze proteins for 24 hours at -1.3 oC with near 100% organ survival and better viability scores than those stored at 4 oC with tradition UW solution1,2. The viability was scored based on observations at 5, 30, and 90 minutes and 24-hour post-transplantation observation of the recipient rats1.

Figure 1.Ice crystal shapes and AFP concentrations 5. (A) No/Low concentration of AFPs: large ice crystals form from no/limited ice curvature6.(B)Optimal AFP concentration: Type III AFP forms hexagonal bipyramidal crystals the create ice curvature to inhibit growth. (C) High AFP concentration: excessive curvature makes it difficult for water molecules to bind however sharp, needle-like crystals form7 and penetrate cell membranes2.

While overall successful, these experiments introduced another issue to the preservation of organs, one related to osmolarity. As solution freezes with AFPs present, the concentration of solutes outside of the cells becomes more concentrated with decreasing amounts of liquid. The extracellular environment thus becomes hypertonic in relation to the cell interior and water rushes out of the cell causing it to shrink. Upon tissue thawing, large pools of water can cause cells to swell and rupture, resulting in non-viable samples. The optimal concentration of AFPs enables both effective inhibition of ice growth and minimization of AFP-induced cell damage4. Figure 1 demonstrates the variable ice crystal shapes that occur depending on AFP concentration.

The Ice Queen aims to optimize the situations described above. In using Type III AFPs, a solution can be cooled below 0oC enabling longer storage of organs. In attaching these AFPs to a scaffold unit, the concentration of solutes can be controlled to optimize AFP concentration and eliminate a problematic osmotic gradient. In essence, the attachment of AFPs to a scaffold increases the local concentration of active proteins while balancing the discrepancy between the total solute concentrations on either side of the cell membrane.

DESIGN & CLONING

In the planning of the Ice Queen, two key considerations were taken. First, a scaffold unit had to be selected in addition to a method of attachment for the AFPs and scaffold subunits. The singe subunit, T3-10 self-assembling cage (PDB: 4EGG) and the E/K coil system (PDB: 1U0I) were selected for these roles respectively. More information on the selection and modeling performed on the designed structure is provided in Background and Modeling.

Type III AFP was selected for use with our protein scaffold because of its moderate activity and well-resolved structure. Minimal cysteine residues meant little potential for incorrect disulfide forming, making it ideal for use in recombinant plasmids. The Davies lab graciously provided us with the QAE isoform (PDB: 1AME) of the antifreeze protein in a pET20-b vector, which has a C-terminal His-tag. While we used ice-affinity purification the His-tag would allow users without access to the ice-affinity purification apparatus to purify the protein in a single step (BBa_K1831001). We also created a variation on the construct without a His-tag (BBa_K1831003), which can be purified using the ice affinity technique. This practice enables simple separation of the AFP from any His-tagged protein, such as our T3-10 scaffold construct (BBa_K1831002). More information of the BioBricks can be found on the Parts page.

AFP-E coil

The Type III AFP was acquired in a pET vector with a XhoI site at the C-terminus of the protein and immediately before the C-terminal His-tag. Oligonucleotide sequences for the E coil were designed and ordered for insertion into the XhoI site. The constructed vector design is shown in Figure 2.

Figure 2.AFP-E coil construct design.

The E coil was successfully cloned into the XhoI site and sequencing confirmed that the coil was inserted in the correct orientation.

Scaffold-K coil

The T3-10 was received in a pET29b vector with a His-tag following the XhoI site (at the C-terminus of the scaffold subunit). Similar to the AFP – E coil, the insert with the K coil was designed to be incorporated into the XhoI site. The oligonucleotide sequences were ordered with the addition of a BamHI site (translated to Gly-Ser) for easy identification of the insert. The vector did not contain any other BamHI restriction sites. The constructed vector design is shown in Figure 3.

Figure 3.Scaffold-K coil construct design.

Difficulties arose in the cloning of the K coil into the XhoI site. The construct was instead ordered as a linear double stranded DNA piece with the construct design shown in Figure 4. This construct varied slightly from the original design to eliminate the second XhoI site and add a His-tag within the restriction sites to enable insertion into multiple vectors.

Figure 4 Re-designed scaffold-K coil construct.

PROTEIN EXPRESSION & PURIFICATION

AFP-E coil

The modified Type III AFP was expressed and purified using ice affinity purification. This technique was first developed in 2003 at Queen’s University8 uses the ability of AFPs to bind to an ice shell to separate the protein from other lysate components. The exploitation of this property enables simultaneous purification and activity confirmation of an antifreeze protein.

Two rounds of ice affinity purification were performed with the AFP – E coil construct with the liquid fraction representing the components not incorporated into the ice shell and the ice fraction the thawed ice shell from each round. The results of these rounds are shown in Figure 5. Round 1 saw the incorporation of number of non-specific components. However, the second round of purification saw the desired protein as the predominant inclusion and provided sufficient purification for experimental purposes. Figure 6 demonstrates the size difference between the AFP-E coil and the wild-type AFP.

Figure 5. Ice affinity purification of AFP-E coil.The supernatant represents the pre-shell supernatant lysate from protein expression. The first round of ice affinity purification included the incorporation of a number of components into the ice fraction. The second round of purification left the predominant component as the AFP with the attached E coil.
Figure 6. Visualization of the slight difference in size between the wild-type AFP and the AFP-Ecoil.


























Scaffold-K coil

While time restraints prevented the expression of the scaffold with the added coil, the expression of the scaffold unit itself was explored. Expression according to the Baker/Yeates protocol9 showed high protein expression rates and confirmed subunit size. The His-tag on the C-terminus was used to purify the protein using a Ni2+ column. The result of the purification steps is shown in Figure 6. E1, E2 and E3 were combined and used for determination of assembly size.

Figure 6. Unmodified T3-10 SDS-PAGE gel.The entire protocol was performed to check expression, purification, and assembly of the scaffold.
Figure 7. Unmodified T3-10 size exclusion chromatogram.

The purified elution fractions were pooled and run through an S200 size exclusion column. The molecular weight of each subunit, calculated from the protein sequence9 is 22 kDa. The T3-10 scaffold is expected to assembly into a 12-mer unit with a minor portion as 9-mer units. Thus, for the S200 column used, the fully and partially assembled scaffolds are predicted to elute between 60-64 mL. The resultant chromatogram is shown in Figure 7. The results indicate that the majority of the protein is assembled in trimer state. Optimization of the assembly conditions is therefore required upon expression of the scaffold with the coil. This will be explored at a later time.

REFERENCES

1. Amir, G., B. Rubinsky, S. Y Basheer, L.Horowitz, L. Jonathan, M. S. Feinberg, A. K. Cmolinky and J. Lavee. (2005). “Improved viability and reduced apoptosis in sub-zero 21-hour preservation of transplanted rat hearts using antifreeze proteins”. The Journal of Heart and Lung Transplantation. 24(11): 1915 – 1929

2. Amir, G., B. Rubinsky, L. Horowitz, L. Miller, J. Leor, Y. Kassif, D. Mishaly, A. Smolinsky and J. Lavee. (2004). “Prolonged 24-hour subzero preservation of heterotopically transplanted rat hearts using antifreeze proteins derived from arctic fish”. Ann Thorac Sugr. 77: 1648 – 1655

3. Fremes, S.E., R. K. Li, R. D. Weisel, D. A. Mickle and L. C. Tumiati. (1991). “Prolonged hypothermic cardiac storage with University of Wisconsin solution: an assessment with human cell cultures”. J Thorac Cardiovasc Surg. 102(5): 666 – 672

4. Carpenter, J.F. and T.N. Hansen. (992). “Antifreeze protein modulates cell survival during cryopreservation: Mediation through influence on ice crystal growth”. Proc. Natl. Acad. Sci. USA. 89:8953–8957

5. Davies, P. L. and C. L. Hew. (1990). “Biochemistry of fish antifreeze proteins”. FASEB Journal. 4(8): 2460 – 2468

6. DeLuca, C. I., R. Comley, P. L. Davies. (1998). “Antifreeze proteins bind independently to ice”. Biophysical Journal. 74(3): 1502 - 1508

7. Knight. C. A., A. L. De Vries and L. D. Oolman. (1984). “Fish antifreeze protein and the freezing and recrystallization of ice”. Nature. 308: 295 – 296

8. Kuiper, M. J., C. Lankin, S. Y. Gauthier, V. K. Walker and P. L. Davies. (2003). “Purification of antifreeze proteins by adsorption to ice. Biochemical and Biophysical Research Communications”. 300: 645 – 648

9. King, N. P., W. Sheffler, M. R. Sawaya, B. S. Vollmar, J. P. Sumida, I. André, T. Gonen. T. O. Yeates an D. Baker. (2012). “Computational design of self-assembling protein nanomaterials with atomic level accuracy”. Science 336(6085): 1171 - 1174