Methods in Biosynthesis and Characterization of the Antifreeze Protein (AFP) for Potential Blood Cryopreservation

The cryopreservation of red blood cells (RBCs) is very important to modern medicine. Cryoprotectants (CPAs) such as dimethyl sulfoxide (DMSO), proline, trehalose, and polyvinyl alcohol (PVA) have been used in the cryopreservation of RBCs, but the results are not satisfactory. Marinomonas primoryensis antifreeze protein (MpAFP) is a Ca2+-dependent AFP derived from Antarctic bacteria, which can prevent bacteria from freezing under extremely cold conditions and may be suitable for cryopreservation of RBCs. The active region of MpAFP is located in region IV and is called MPAFP_RIV. In this paper, the gene of region IV of MpAFP is introduced into BL21 (DE3) competent cells, and MpAFP_RIV is obtained after culture, separation, and purification. The improved splat assay is proposed, and this method first proves that MpAFP_RIV has strong ice recrystallization inhibition (IRI) activity without Ca2+. The improved splat assay is easier to operate and lower the cost compared to the traditional one, and the results are consistent with the classic sucrose sandwich assay, proving that this method can accurately detect IRI activity. The feasibility of MPAFP_RIV combined with classical CPA for cryopreservation of RBCs and the methods to increase the yield of MPAFP_RIV are proposed.

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The effect of surface charge on the thermal stability and ice recrystallization inhibition activity of antifreeze protein III (AFP III)

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2017.11.073 ◽

2018 ◽

Vol 495 (1) ◽

pp. 1055-1060 ◽

Cited By ~ 6

Author(s):

R.C. Deller ◽

B.M. Carter ◽

I. Zampetakis ◽

F. Scarpa ◽

A.W. Perriman

Keyword(s):

Thermal Stability ◽

Surface Charge ◽

Antifreeze Protein ◽

Inhibition Activity ◽

Ice Recrystallization Inhibition ◽

Ice Recrystallization ◽

Recrystallization Inhibition ◽

Effect Of Surface

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Prospecting for ice association: characterization of freeze–thaw selected enrichment cultures from latitudinally distant soils

Canadian Journal of Microbiology ◽

10.1139/w2012-010 ◽

2012 ◽

Vol 58 (4) ◽

pp. 402-412 ◽

Cited By ~ 9

Author(s):

Sandra L. Wilson ◽

Paul Grogan ◽

Virginia K. Walker

Keyword(s):

Microbial Consortia ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Ice Recrystallization Inhibition ◽

Ice Recrystallization ◽

Freeze Thaw ◽

Recrystallization Inhibition ◽

And Function ◽

Selective Regime

Freeze–thaw stress has previously been shown to alter soil community structure and function. We sought to further investigate this stress on enriched microbial consortia with the aim of identifying microbes with ice-associating adaptations that facilitate survival. Enrichments were established to obtain culturable psychrotolerant microbes from soil samples from the latitudinal extremes of the Canadian Shield plateau. The resulting consortia were subjected to consecutive freeze–thaw cycles, and survivors were putatively identified by their 16S rRNA gene sequences. Even though the northerly site was exposed to longer, colder winters and large spring-time temperature fluctuations, the selective regime similarly affected both enriched consortia. Quantitative PCR and metagenomic sequencing were used to determine the frequency of a subset of the resistant microbes in the original enrichments. The metagenomes showed 22 initial genera, only 6 survived and these were not dominant prior to selection. When survivors were assayed for ice recrystallization inhibition and ice nucleation activities, over 60% had at least one of these properties. These phenotypes were not more prevalent in the northern enrichment, indicating that regarding these adaptations, the enrichment strategy yielded seemingly functionally similar consortia from each site.

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