Several types of
natural molecules interact specifically with ice
crystals. Small antifreeze proteins (AFPs) adsorb to particular facets
of ice crystals, thus inhibiting their growth, whereas larger ice-nucleating
proteins (INPs) can trigger the formation of new ice crystals at temperatures
much higher than the homogeneous ice nucleation temperature of pure
water. It has been proposed that both types of proteins interact similarly
with ice and that, in principle, they may be able to exhibit both
functions. Here we investigated two naturally occurring antifreeze
proteins, one from fish, type-III AFP, and one from beetles, TmAFP. We show that in addition to ice growth inhibition,
both can also trigger ice nucleation above the homogeneous freezing
temperature, providing unambiguous experimental proof for their contrasting
behavior. Our analysis suggests that the predominant difference between
AFPs and INPs is their molecular size, which is a very good predictor
of their ice nucleation temperature.
Ice-binding proteins (IBPs) permit their hosts to thrive in the presence of ice. The ability of IBPs to control ice growth makes them potential additives in industries ranging from food storage and cryopreservation to anti-icing systems. For IBPs to be used in commercial applications, however, methods are needed to produce sufficient quantities of high-quality proteins. Here, we describe a new method for IBP purification, termed falling water ice affinity purification (FWIP). The method is based on the affinity of IBPs for ice and does not require molecular tags. A crude IBP solution is allowed to flow over a chilled vertical surface of a commercial ice machine. The temperature of the surface is lowered gradually until ice crystals are produced, to which the IBPs bind but other solutes do not. We found that a maximum of 35 mg of IBP was incorporated in 1 kg of ice. Two rounds of FWIP resulted in >95% purity. An ice machine that produces 60 kg of ice per day can be used to purify one gram of IBP per day. In combination with efficient concentration of the protein solution by tangential flow filtration the FWIP method is suitable for the purification of grams of IBPs for research purposes and applications.
15The temperature of the surface is lowered gradually until ice crystals are produced, to which the
16IBPs bind but other solutes do not. As in other ice affinity methods, FWIP does not require 17 molecular tags and is suitable for purifying recombinant IBPs as well as IBPs from natural sources.
18The advantage of FWIP over other ice affinity methods is that it exploits an ice machine designed to
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