抗冻蛋白研究进展

抗冻蛋白是一类具有热滞效应、冰晶形态效应和重结晶抑制效应的蛋白质。简单介绍了各种抗冻蛋白的生化特征、作用机制及其应用研究 ,并对抗冻蛋白的基因和基因工程研究作了较为系统的综述。     

抗冻蛋白及其在植物抗冻基因工程的应用

从应用的角度系统综述了抗冻蛋白（ＡＦＰs)的特性、活性、用途、生化特征、在细菌中的表达,在植物抗冻生理中的作用及其基因工程,简洁地讨论了抗冻蛋白的研究现状和最新进展。     

鱼类抗冻蛋白的研究进展

抗冻蛋白 (AFP)可非依数性地降低溶液冰点 ,对冷冻细胞和胚胎具有高效的保护作用。目前的研究表明 ,不同的鱼类抗冻蛋白尽管都具有降低冰点的活性 ,但在结构和组成上又存在有较大的差异。根据其结构和化学组成 ,一般将它们分为 4大类 :AFP I、AFP II、AFP III和AFP IV。抗冻蛋白的编码基因为基因组中多拷贝基因家族的成员 ,其基因表达在很大程度上要受到季节变化的影响。目前 ,普遍使用吸附抑制假说来解释AFP非依数性降低溶液冰点的分子机制 ,但不同类抗冻蛋白在降低溶液冰点时的作用模式却不尽相同。现就鱼类的 4类抗冻蛋白的结构组成、基因性质、抗冻机制及其在细胞和胚胎冻存中的作用等领域的研究进展进行概括性综述     

抗冻蛋白及其在植物抗冻生理中的作用

抗冻蛋白（ａｎｔｉｆｒｅｚｅｐｒｏｔｅｉｎｓ,ＡＦＰｓ）是一类抑制冰晶生长的蛋白质,能以非依数性形式降低水溶液的冰点,但对熔点影响甚微,从而导致水溶液的熔点和冰点之间出现差值。这种差值称为热滞活性（ｔｈｅｒｍａｌｈｙｓｔｅｒｅｓｉｓａｃｔｉｖｉｔｙ,...     

赤翅甲抗冻蛋白基因的原核表达及蛋白生物活性检测

根据GenBank中序列人工合成赤翅甲Dendroides canadensis的抗冻蛋白基因（afp）,将其克隆到载体pGEX-4T-1上,构建融合表达的重组质粒,转化大肠杆菌 BL21并进行原核表达。通过优化表达的诱导条件和SDS-PAGE检测,证明人工合成的赤翅甲抗冻蛋白基因能够特异性地表达,并以可溶性融合蛋白形式存在,相对分子质量约为40 kD。抗冻蛋白的生物活性检测表明,赤翅甲的抗冻融合蛋白能够提高细菌的耐寒能力。     

植物抗冻蛋白及抗冻性分子改良

概述了植物抗冻蛋白及其相关基因的研究现状,主要包括植物低温诱导蛋白、具有热滞活性的植物抗冻蛋白及其相关基因的分离、鉴定与表达调控,以及植物抗冻性基因工程研究动态.在此基础上,讨论了该领域研究中的主要问题、发展趋势及近期研究热点.     

植物抗冻蛋白研究进展

抗冻蛋白(AFPs)最初是从极区海鱼中发现的一种适应低温的特异蛋白质, 它能阻止体液内冰核的形成与生长,维持体液的非冰冻状态.对近年来植物AFPs的发现过程,AFP的生化特性,抗冻作用机制,抗冻蛋白基因工程及其应用前景等作了系统的综述.     

昆虫抗冻蛋白的分离纯化及特性分析

昆虫抗冻蛋白具有很高的热滞活性,可保护机体免受结冰引起的伤害。昆虫抗冻蛋白的分离纯化多采用凝胶过滤层析、离子交换层析及HPLC等技术,已用于鱼类抗冻蛋白纯化的冰亲和纯化(IAP)技术也可考虑应用于昆虫抗冻蛋白的分离提纯。昆虫抗冻蛋白具有高活性,规则的一级结构及类似的冰晶结合表面等特性。     

The mechanism by which fish antifreeze proteins cause thermal hysteresis

Antifreeze proteins are characterised by their ability to prevent ice from growing upon cooling below the bulk melting point. This displacement of the freezing temperature of ice is limited and at a sufficiently low temperature a rapid ice growth takes place. The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. This causes the ice to grow as convex surface regions between adjacent adsorbed antifreeze proteins, thus lowering the temperature at which the crystal can visibly expand. The model requires that the antifreeze proteins are irreversibly adsorbed onto the ice surface within the hysteresis gap. This presupposition is apparently in conflict with several characteristic features of the phenomenon; the absence of superheating of ice in the presence of antifreeze proteins, the dependence of the hysteresis activity on the concentration of antifreeze proteins and the different capacities of different types of antifreeze proteins to cause thermal hysteresis at equimolar concentrations. In addition, there are structural obstacles that apparently would preclude irreversible adsorption of the antifreeze proteins to the ice surface; the bond strength necessary for irreversible adsorption and the absence of a clearly defined surface to which the antifreeze proteins may adsorb. This article deals with these apparent conflicts between the prevailing theory and the empirical observations. We first review the mechanism of thermal hysteresis with some modifications: we explain the hysteresis as a result of vapour pressure equilibrium between the ice surface and the ambient fluid fraction within the hysteresis gap due to a pressure build-up within the convex growth zones, and the ice growth as the result of an ice surface nucleation event at the hysteresis freezing point. We then go on to summarise the empirical data to show that the dependence of the hysteresis on the concentration of antifreeze proteins arises from an equilibrium exchange of antifreeze proteins between ice and solution at the melting point. This reversible association between antifreeze proteins and the ice is followed by an irreversible adsorption of the antifreeze proteins onto a newly formed crystal plane when the temperature is lowered below the melting point. The formation of the crystal plane is due to a solidification of the interfacial region, and the necessary bond strength is provided by the protein "freezing" to the surface. In essence: the antifreeze proteins are "melted off" the ice at the bulk melting point and "freeze" to the ice as the temperature is reduced to subfreezing temperatures. We explain the different hysteresis activities caused by different types of antifreeze proteins at equimolar concentrations as a consequence of their solubility features during the phase of reversible association between the proteins and the ice, i.e., at the melting point; a low water solubility results in a large fraction of the proteins being associated with the ice at the melting point. This leads to a greater density of irreversibly adsorbed antifreeze proteins at the ice surface when the temperature drops, and thus to a greater hysteresis activity. Reference is also made to observations on insect antifreeze proteins to emphasise the general validity of this approach.     

Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. Theafa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.     

昆虫抗冻蛋白的研究进展

热滞效应(Tberm Hysteresis Action)最早在昆虫研究中发现,后来研究表明,它是抗冻蛋白(Antifreeze Proteins,AFPs)的一种基本性质。和鱼类,植物AFPs相比,昆虫AFPs具有更高 的热滞活性和独特的化学结构特征。昆虫AFPs在昆虫抗冻生理过程中起着相当重要的作用,表现在以下三个方面：①抑制一些冰晶形成;②提高冰冻耐受性;③可能参与水分平衡过程。光周期,气温和湿度是调控AFPs生物合成与降解的三种外部因子,而体内激素的变化可能是直接调节脂肪体合成AFPs的内部因子。     

Structure prediction of membrane proteins

There is a large gap between the number of membrane protein (MP) sequences and that of their decoded 3D structures, especially high-resolution structures, due to difficulties in crystal preparation of MPs. However, detailed knowledge of the 3D structure is required for the fundamental understanding of the function of an MP and the interactions between the protein and its inhibitors or activators. In this paper, some computational approaches that have been used to predict MP structures are discussed and compared.     

植物抗冻蛋白研究进展(综述)

植物抗冻蛋白(AFPs)的研究起步较晚.本文综述植物AFPs的发现过程、理化性质、在植物抗冻生理中的作用及其机制、结构模型及相关的植物基因工程.     

Distinct molecular features facilitating ice-binding mechanisms in hyperactive antifreeze proteins closely related to an Antarctic sea ice bacterium

Antifreeze proteins or ice-binding proteins (IBPs) facilitate the survival of certain cellular organisms in freezing environment by inhibiting the growth of ice crystals in solution. Present study identifies orthologs of the IBP of Colwellia sp. SLW05, which were obtained from a wide range of taxa. Phylogenetic analysis on the basis of conserved regions (predicted as the ‘ice-binding domain’ [IBD]) present in all the orthologs, separates the bacterial and archaeal orthologs from that of the eukaryotes’. Correspondence analysis pointed out that the bacterial and archaeal IBDs have relatively higher average hydrophobicity than the eukaryotic members. IBDs belonging to bacterial as well as archaeal AFPs contain comparatively more strands, and therefore are revealed to be under higher evolutionary selection pressure. Molecular docking studies prove that the ice crystals form more stable complex with the bacterial as well as archaeal proteins than the eukaryotic orthologs. Analysis of the docked structures have traced out the ice-binding sites (IBSs) in all the orthologs which continue to facilitate ice-binding activity even after getting mutated with respect to the well-studied IBSs of Typhula ishikariensis and notably, all these mutations performing ice-binding using ‘anchored clathrate mechanism’ have been found to prefer polar and hydrophilic amino acids. Horizontal gene transfer studies point toward a strong selection pressure favoring independent evolution of the IBPs in some polar organisms including prokaryotes as well as eukaryotes because these proteins facilitate the polar organisms to acclimatize to the adversities in their niche, thus safeguarding their existence.     

Cloning, sequencing and prokaryotic expression of cDNAs for antifreeze protein family from beetle Tenebrio molitor

Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-l-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3- tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blot-ting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the founda-tion for further studies on the properties and functions of insect antifreeze proteins.