Description:
Recombinant type III antifreeze protein, also called ice-binding protein, thermal hysteresis protein, and ice-structuring protein, rQAE m1.1 isoform from ocean pout (Macrozoarces americanus), produced in E. coli and purified by Ni-NTA affinity chromatography. The protein contains 66 amino acids with an addition of a 6xHistidine tag at its C-terminus for affinity purification. The molecular weight of the complete protein is 8.1 kDa. The native QAE isoform, also called HPLC 12, is one of at least 12 different isoforms isolated from serum of ocean pout. The rQAE m1.1 isoform is a recombinant gene that closely matches the native QAE sequence. The differences between the rQAE m1.1 and the native QAE are in the initiating methionine and the terminating YAA that replaces the native YPPA. This c terminal mutation was required for protein stability and was shown to be indistinguishable from native QAE in its antifreeze activity
(Chao, H., Davies, P. L., Sykes, B. D. & Sönnichsen, F. D. Protein Sci. 2, 1411–1428, 1993;
Chao, H., Sönnichsen, F. D., DeLuca, C. I., Sykes, B. D. & Davies, P. L. Protein Sci. 3, 1760–1769, 1994).
Source:
E. coli
Physical Appearance:
White lyophilized (freeze-dried) powder.
Formulation:
The protein was lyophilized from a concentrated (0.8 mg/ml) solution with 0.4 mg/ml of NaHCO3
Solubility:
It is recommended to reconstitute the lyophilized AFP in 0.4% NaHCO3 or water, not less than 100µg/ml, which can then be further diluted to other aqueous solutions, preferably in a presence of a carrier protein such as BSA or similar.
Stability:
Lyophilized AGF although stable at room temperature for at least 4 weeks, should be stored desiccated below -18C. Upon reconstitution and filter sterilization AGF can be stored at 4C, pH 8 for up to 4 weeks. For long term storage and more diluted solutions it is recommended to add a carrier protein (0.1% HSA or BSA).
Purity:
Greater than 98.0% as determined by:
(a) Gel filtration at non denaturing conditions using 25 mM Tris-HCl + 150 nM NaCl, pH 8.
(b) Analysis by reducing and non-reducing SDS-PAGE gel.
Amino Acid Sequence:
AAQASVVANQ LIPINTALTL VMMRSEVVTP VGIPAEDIPR LVSMQVNRAV PLGTTLMPDM VKGYAAKDEL HHHHHH.
Dimers and Aggregates:
Less than 4% as determined by gel filtration chromatography.
Biological Activity:
The AFPIII(rQAE) has a strong recrystallization inhibition activity (at the micromolar level, 0.01 mg/ml is sufficient for effective recrystallization inhibition, Budke et. al. Cryst. Growth Des., 14, 9, 4285–4294, 2014). The typical thermal hysteresis activity (TH) of recombinant ocean pout type III antifreeze protein rQAE m1.1 is TH= 0.029 o C *sqrt (concentration in micromolar) For example: 0.5 o C at 0.3 mM (Chao, H., Davies, P. L., Sykes, B. D. & Sönnichsen, F. D. Protein Sci. 2, 1411–1428, 1993). The protein retains its full activity in a pH range of 2 to 11 and it is stable at room temperature and below (Chao, H., Sönnichsen, F. D., DeLuca, C. I., Sykes, B. D. & Davies, P. L. Protein Sci. 3, 1760–1769, 1994).a
Endotoxin:
Less than 0.05 ng/µg (0.5 EU/µg)
Protein content:
Protein quantitation was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.18 as the extinction coefficient for a 0.1% (1mg/ml) solution. This value is calculated by the DNAman computer analysis program of protein sequences.
Usage:
For research only
