Antifreeze proteins (AFPs) are a class of proteins that enable organisms such as fishes, plants and insects to survive in sub-zero environments by lowering the freezing point in a non-colligative manner (Bar, et al., 2006). The AFPs bind to ice and inhibit the recrystallisation process that is lethal to organisms.AFPs have varying structures and sequences although they have the same function (Bar, et al., 2006). These factors play a critical role in their functions. These could also be the plausible reasons why insect AFPs, with І-helix structures and numerous disulfide bonds, have higher specific activity than fish AFPs.
As a result, insect AFPs are being studied on their potential uses in various industries. One example is the Tenobrio molitor AFP (TmAFP), noted to be threonine- and cysteine-rich with eight disulfide bonds (Bar, et al., 2006). As industrial applications may involve large amount of TmAFP, they would also have to be produced in sufficient quantity. Therefore, the different expression and purification systems will be covered in this literature review.
There have been previous attempts in expressing native TmAFP in Escherichia coli, some were successful while others encountered difficulties such as non-functional TmAFP being produced due to absence of disulfide bonds or over-expressed TmAFP forming inclusion bodies (Bar, et al., 2006). Currently, researchers have experimented with different vectors, E.coli strains and even yeast to synthesise properly folded, active TmAFP.a. Escherichia coliBar, et al. (2006) transformed various recombinant TmAFP constructs into E.coli Origami B (DE3) plysS strain. This particular strain has a mutation that promotes the formation of disulfide bond, essential to the structure of TmAFP.
All of the fusion proteins were over-expressed at low temperature (15oc) which was shown to have assisted in the folding of TmAFP and the formation of disulfide bonds (Bar, et al., 2006). Compared to previous experiments, in-vitro folding was not required, which was an extensive and labour-intensive process (Bar, et al., 2006).
To demonstrate that the recombinant TmAFPs expressed were active and correctly folded, they were subjected to different tests. The 5,5-dithiobis(2-nitrobenzoic acid) assay which is used to quantify free thiol groups in protein samples revealed no free thiol group, suggesting that disulfide bonds were successfully formed while the thermal hysteresis (TH) activity of the recombinant TmAFP was similar to previous works on active TmAFP (Bar, et al., 2006). However, there were still incorrectly folded TmAFPs being expressed, though they were removed by purification.b. Pichia pastorisP.pastoris is a yeast that is able to express large quantity of recombinant proteins. In an experiment by Tyshenko, et al. (2006), they transformed recombinant vector TmAFP-pPIC9 into three strains, GS115, KM71, and X33. Afterwards, they were subjected to small- and large-scale fermentation. Surprisingly, there was no TH activity observed for the recombinant TmAFP although ice recrystallisation was inhibited. It was suggested that TmAFPs underwent glycosylation in P.pastoris and interfered with their ability to lower freezing temperature (Tyshenko, et al., 2006). Hence, it would be possible to conclude P.pastoris may not be suitable for expressing TmAFP even though larger amounts are synthesised compared to E.coli.
Affinity chromatography takes advantage of bio-specific binding interaction between immobilised ligands on the stationary phase and analytes to purify the analyte. For example, in immobilised metal affinity chromatography, polyhistidine tagged proteins bind to metal ligands like nickel while contaminants are not adsorbed and be eluted as dead volume. In the same experiment by Bar, et al. (2006)
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