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CN-121975019-A - Design of novel antifreeze polypeptide and protection method for cell cryopreservation

CN121975019ACN 121975019 ACN121975019 ACN 121975019ACN-121975019-A

Abstract

The invention discloses a novel antifreeze polypeptide with definite structure and excellent performance and a method for protecting frozen cells. The core structure of the polypeptide is formed by covalently connecting two functional modules, wherein the N end is a unit with ice crystal binding property, the C end is a super-hydrophilic unit, and the two units are bridged by a flexible linker. Experiments prove that the synthetic polypeptide has excellent anti-freezing activity, can obviously inhibit the nucleation, growth and recrystallization processes of ice crystals, and has particularly outstanding ice recrystallization inhibition activity. The novel antifreeze polypeptide exhibits excellent biocompatibility and is suitable for low-temperature cryopreservation of cells and other living microbial preparations sensitive to freezing damage. The result shows that the polypeptide can greatly improve the freeze-drying survival rate and the activity after resuscitating of cells, has no toxicity to cells, and has the potential of replacing the traditional cryoprotectant. Therefore, the invention has wide application prospect in the low-temperature preservation technology of food industry, biological medicine, agricultural breeding and biological sample library.

Inventors

  • QI HAISHAN
  • CUI ZHONGXIN

Assignees

  • 天津大学

Dates

Publication Date
20260505
Application Date
20260126

Claims (10)

  1. 1. A polypeptide having anti-freeze activity, wherein the polypeptide comprises a first functional fragment and a second functional fragment, wherein the first functional fragment is an ice crystal binding module, and the second functional fragment is an ultra-hydrophilic module.
  2. 2. The polypeptide of claim 1, wherein the ice crystal binding moiety and the superhydrophilic moiety are linked by a flexible linker peptide.
  3. 3. A method of constructing the polypeptide of claim 1 or 2, comprising the steps of: S1, screening key ice crystal binding modules including but not limited to GE, GT, TST and the like; s2, designing super-hydrophilic modules, including but not limited to NNT, NNE, EKT, ETK and the like; s3, combining the two functional fragments by using a flexible linker, including but not limited to GS 4 、G 2 S 3 、G 3 S 2 and the like.
  4. 4. The polypeptide according to claim 1 or 2, characterized in that it has anti-freeze properties.
  5. 5. The novel antifreeze polypeptide of claim 4, wherein the concentration of 0.1 to 1% of the novel antifreeze polypeptide has an IRI of 10 to 20%.
  6. 6. The novel antifreeze polypeptide of claim 4, wherein the ice crystal growth rate at supercooling degree of-0.3℃is from 6 to 8. Mu.m/s.
  7. 7. The novel antifreeze polypeptide of claim 4, wherein the novel antifreeze polypeptide has a freezing point depression of from 2 to 5℃at a concentration of from 0.1 to 1%.
  8. 8. Use of the novel antifreeze polypeptide of claim 4 as a cell cryopreservation protector.
  9. 9. The method according to claim 8, wherein the cells are subjected to serial subculture and then frozen at low temperature by adding novel antifreeze polypeptide (0.1-1% concentration).
  10. 10. The method of claim 8, wherein the cells comprise probiotics, erythrocytes, hematopoietic stem cells, sperm cells, egg cells, etc., and have a viability of greater than 90%.

Description

Design of novel antifreeze polypeptide and protection method for cell cryopreservation Technical Field The invention relates to the technical field of microorganisms, in particular to a design and construction method and application of novel antifreeze polypeptide. Background Cryopreservation is a key technology for preserving active microbial materials such as cells for a long period of time. However, during the freezing and thawing process, the formation, growth and recrystallization of ice crystals can cause mechanical damage to biological macromolecules such as cell membranes, proteins and the like, and cause severe changes in osmotic pressure of the solution, resulting in dehydration and rupture of cells, and severely reducing the survival rate and bioactivity of the cells. Therefore, developing a high-efficiency and safe cryopreservation protective agent is important for maintaining the activity of cells and guaranteeing the application efficacy of the cells. The commonly used cryopreservation protective agents mainly comprise micromolecular osmotic protective agents (such as glycerol and dimethyl sulfoxide) and macromolecular non-osmotic protective agents (such as sucrose, polyvinylpyrrolidone and serum albumin). The micromolecular protective agent can permeate cells to reduce the freezing point, but can have cytotoxicity and influence on subsequent metabolic activity, and the macromolecule protective agent mainly forms a glassy matrix outside the cells to inhibit ice crystal growth, but has limited freezing resistance activity and has biocompatibility problem for partially synthesizing macromolecules. In nature, some organisms (such as polar fish, insects and plants) are adapted to severe cold environments, and can express antifreeze proteins or active fragments thereof with unique ice crystal binding and inhibiting functions. The biological molecules can be reversibly and specifically adsorbed on the surface of ice crystals to inhibit the growth and recrystallization of the ice crystals by the Kelvin effect, so that the ice crystal morphology is effectively modified and mechanical damage is reduced while the freezing point is not reduced quantitatively. Compared with the traditional chemical protectant, the anti-freeze protein/polypeptide has the advantages of biodegradability, strong targeting property, low dosage, good potential cell compatibility and the like, and is regarded as an ideal candidate of a new generation of biological source cryoprotectant. However, the direct use of natural antifreeze proteins presents many challenges, including firstly, limited sources, high extraction and purification costs, secondly, relatively high molecular weight, and the possible risk of immunogenicity, and thirdly, the complete natural structure may not be completely necessary for antifreeze activity, and even partial domains may introduce instability factors. Although studies have been made to simulate the core active fragment of a natural antifreeze protein by genetic engineering expression or chemical synthesis, the designed polypeptide often suffers from single antifreeze activity, insufficient stability or lack of sufficient hydrophilicity to function effectively at the ice-water interface. Aiming at application scenes such as cryopreservation of probiotics, erythrocytes and hematopoietic stem cells, the polypeptide is required to be capable of inhibiting ice crystals in a complex matrix with high efficiency, and also capable of well interacting with the cell surface to provide more comprehensive protection. Therefore, there is a need in the art to develop a novel antifreeze polypeptide that has a simple structure, high activity, easy synthesis, and optimal design for cell cryopreservation requirements. By means of rational design strategy, the novel antifreeze polypeptide with high-efficiency antifreeze performance is constructed, the limitation of the prior art is hopeful to be broken through, and a safer, high-efficiency and specific solution is provided for the low-temperature preservation of cells and other bioactive preparations. Disclosure of Invention The invention aims to provide a design method of novel antifreeze polypeptide. Another technical problem to be solved by the present invention is to provide the application of the novel antifreeze polypeptide. In order to solve the technical problems, the technical scheme of the invention is as follows: a novel antifreeze polypeptide is prepared from the new components of polypeptide. The construction method of the novel antifreeze polypeptide comprises the following specific steps: S1, screening and optimizing core ice crystal combination module Based on the consensus sequence and spatial conformation of ice crystal binding surfaces of known antifreeze proteins, bioinformatics tools are used to screen the smallest active units with high binding affinity. The combination stability of the molecular dynamics simulation-based polymer and the primary