Lin, F.H.; Sun, T.J.; Fletcher, G.L.; Davies, P.L. (2012). Thermolabile antifreeze protein produced in Escherichia coli for structural analysis. Protein Expression and Purification. 82 (1) 75-82.
The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforrns; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express (R) strain of Escherichia coil at 12 degrees C, just below the thermal denaturation temperature of 16-18 degrees C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP 111 yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes for the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography. (C) 2011 Elsevier Inc. All rights reserved.