蛋白质内含肽及其生物学意义

蛋白质内含肽是存在于前体蛋白质中的一段多肽链,靠自我剪切的方式从前体蛋白中释放出来。蛋白质内含肽的发现,不仅在理论上丰富了遗传信息翻译后加工的内容,而且在实践上有重大的生物学意义,特别是在蛋白质纯化方面有着广泛的应用前景。本文就蛋白质内含肽的发现、特征、鉴定、剪接机制及其生物学意义作一概述。     

内含肽介导的蛋白连接技术及其应用

内含肽介导的蛋白质剪接是一种自发的翻译后事件,内含肽可介导其自身从前体蛋白上切除,同时将其两侧的外显肽连接起来.在过去10多年中,基于蛋白质剪接原理发展出的蛋白连接技术被广泛的用于蛋白质工程的研究中.这些技术打破了化学合成方法中对目标物大小的限制,有助于化学和生物学的研究.针对近年由蛋白质连接演化出来的新技术及其应用做简要的阐述.     

微生物内含肽在基因治疗中的应用

蛋白质内含肽是存在于前体蛋白质中的一段多肽,依靠蛋白质自剪接这一特殊机制从前体蛋白中释放出来,并且使两侧的蛋白质外显肽连接成为成熟的蛋白质。本文就内含肽在基因治疗方面的研究做一综述。     

内含肽在构建亲和纯化系统中的应用

内含肽是前体未成熟蛋白中的一段具有自我剪接功能的多肽链,在蛋白质纯化、蛋白质连接、环肽制备、蛋白标记以及生物传感器等方面广泛应用。本文综述了内含肽应用于蛋白质亲和纯化的发展历程,分别对层析型和非层析型内含肽纯化体系进行了分析和讨论,并总结了对控制内含肽断裂反应所进行的研究,为进一步改善内含肽介导蛋白质纯化提供依据和线索。     

蛋白质内含肽的研究及应用进展

内含肽的发现与研究只经过短短的20年,但已在各个生物领域展现出应用潜力。本文阐述了蛋白质内含肽的相关概念、种类、分布、结构、功能和自我剪接机制。分析了内含肽在当前生物技术领域中的应用情况以及存在的优点和局限性,最后就内含肽的应用前景进行了展望。     

内含肽介导的生物学效应及其应用

蛋白质翻译产物在成熟过程中剪切释放出来的一段氨基酸序列称为“intein”---即内含肽。它与前体蛋白以框内融合的形式共同翻译,并内嵌于前体蛋白序列中。内含肽的解离以及内含肽两侧氨基酸序列的连接是在内含肽自身催化作用下完成的。本文将从内含肽的发现、结构特征和作用机理等方面对这种具有特殊意义的蛋白质成熟机制进行较为全面的论述,同时介绍了近年来发展起来的以内含肽介导的蛋白质剪接为基础的蛋白质纯化和改造技术。     

蛋白质剪接研究进展

蛋白质剪接是一个翻译后自催化加工过程,它不需要酶或其他辅助因子的参与。在这个过程中,前体蛋白的Intein(内含肽)被切离,其两侧的Extein(外显肽)连接在一起。Intein按结构可分为经典Intein和微型Intein,其中的经典Intein包括Hint结构域和中间的归巢内切酶结构域(该结构域在微型内含肽中不存在)。蛋白质剪接及其他具有Hint结构域的蛋白加工过程的起始步骤是N-S/O酰基重排反应,该反应是由Hint结构域催化的;Intein的剪接还分为顺式剪接和反式剪接,通过对Intein进行改造,可以阻断剪接过程,但不影响N端肽键或C端肽键的断裂;通过筛选突变体,可以获得温度敏感型、pH敏感型或小分子诱导型的内含肽。这些研究促进了Intein在多肽制备及其它方面的应用。     

蛋白质剪接及其在蛋白质工程中的应用

蛋白质剪接是蛋白质内含肽介导的,一种在蛋白质水平上翻译后的加工过程,它由一系列分子内的剪切-连接反应组成。蛋白质内含肽是一个蛋白质前体中的多肽序列,可以催化自身从蛋白质前体中断裂,使两侧的蛋白质外显肽连接成成熟的蛋白质。蛋白质内含肽的发现,不仅丰富了遗传信息翻译后加工的理论,在实践中也有广泛的应用前景。Abstract: Protein splicing , which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.     

内含肽介导的氯离子通道蛋白CFTR的反式剪接

研究利用内含肽(intein)的蛋白质反式剪接功能在大肠杆菌中对囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane regulator, CFTR)的反式剪接作用.CFTR基因突变导致一种常染色体隐性遗传疾病囊性纤维化(cystic fibrosis, CF).将CFTR的cDNA于剪接反应所需的保守性氨基酸残基Ser-660前断裂为N端和C端,分别与split mini Ssp DnaB 内含肽的106个氨基酸残基的N端和48个氨基酸残基的C端编码序列融合,构建到原核表达载体pBV220 诱导表达后SDS-PAGE可见预期大小剪接形成的CFTR蛋白条带,Western印迹用CFTR特异性抗体进一步证明为剪接所产生的CFTR蛋白,表明内含肽可有效催化CFTR的反式剪接.     

内含肽介导的新型蛋白质纯化技术

内含肽(intein)的自切剖功能在蛋白质工程领域有着广泛的应用,尤其是在蛋白质纯化方面,将内含肽与亲和纯化技术联合应用,使得传统的亲和纯化方式有了重大的突破.近年来,随着内含肽的研究不断深入,一批内含肽介导的新型蛋白质纯化技术不断出现.新技术依靠内含肽的自切割功能去除亲和标签,避免了传统工艺中因外加蛋白酶而带来的诸多麻烦;新的聚合材料的引入又成功地避开了亲和树脂柱的使用,不仅优化了工艺,也大大降低了纯化的成本,为在工业化大生产中的应用奠定了基础.     

PRP8蛋白质反式剪接系统的建立

真菌病原体Cryptococcus neoformansAD血清型剪接体蛋白PRP8蛋白质内含子是目前 发现的第2个存在于真核生物体核基因组中的蛋白质内含子.它的宿主基因prp8编码的PRP 8蛋白作为剪接体的1个组分,是1个高度保守的mRNA剪接蛋白.将组氨酸标签插入克隆自真菌病原体Cryptococcus neoformans AD血清型的PRP8蛋白质内含子中,并将该蛋白质内含子进行人工断裂,获得断裂蛋白质内含子,在大肠杆菌中鉴定其剪接活性.研究结果表明：所获得的改造型蛋白质内含子均表现出高效的剪接活性.利用此Cryptococcus neoformansAD血清型PRP8 断裂蛋白质内含子,成功构建了蛋白质反式剪接系统.这一反式剪接系统可用于其他蛋白质的连接与合成,有望成为蛋白质工程中的一种有用工具.     

断裂蛋白质内含子的剪接机制、起源和进化

蛋白质内含子(intein)是具有自我催化活性的蛋白质. 翻译后,通过蛋白质剪接从蛋白质前体中去掉,并以肽键连接两侧蛋白质外显子(extein)形成成熟蛋白质. 断裂蛋白质内含子(split intein)在蛋白质内含子中部区域特定位点发生断裂,形成N端片段和C端片段,分别由基因组上相距较远的两个基因编码. 现在已知,它仅分布于蓝细菌和古细菌中. 断裂蛋白质内含子的N端片段和C端片段通过非共价键(如静电作用)相互识别,重建催化活性中心,介导蛋白质反式剪接. 断裂蛋白质内含子的发现进一步深化了人们对基因表达和蛋白质翻译后成熟过程复杂性的认识,而且它在蛋白质工程、蛋白质药物开发和蛋白质结构与功能研究等方面有非常广泛的应用. 本文试图综述断裂蛋白质内含子的分布、结构特征和剪接机制,并分析其可能的起源和进化途径.     

蛋白质内含子演化分析

蛋白质内含子是能够自我剪切的一段多肽链 .它在生物三大系统中都存在 ,但它的分布在各物种间以及蛋白质种类之间极不均衡 .蛋白质内含子的演化和扩散也因此引起了人们的极大兴趣 .经过系统搜索 ,从已知的核酸序列中找到 6 9个经典的蛋白质内含子 .同源比较和系统树分析表明 ,蛋白质内含子的演化应综合先天遗传和后天转移两方面的因素 .     

利用ELP-Intein系统在大肠杆菌中生产抗菌肽DLP4

DLP4 （defensin-like peptide 4）是一种新型昆虫防御素抗菌肽,对革兰氏阳性细菌具有强大的抗菌活性而且不易产生抗药性。本研究利用类弹性蛋白（elastin-like polypeptide, ELP）的相变特性和蛋白质内含子（intein, I）的C端断裂系统,通过构建重组表达质粒pET-ELP-I-DLP4,以大肠杆菌（Escherichia coli）作为宿主细胞,诱导表达后的重组蛋白通过简单的离心、pH和温度转变进行纯化得到DLP4。研究中发现,在表达纯化过程中蛋白质内含子发生了C端提前断裂。为了解决这一问题,将其断裂为N端片段（I0_N）和C端片段（I0_C）后,分别与ELP或DLP4融合,构建了pET-ELP-I0_N和pET-ELP-I0_C-DLP4两种重组表达质粒。分别在大肠杆菌中诱导表达,将表达后的菌液混合,使蛋白质内含子恢复C端断裂活性,最终得到的DLP4的得率约为1.49 mg/L。抑菌试验证明纯化的DLP4表现出预期活性,这为DLP4在原核系统中的表达纯化提供...     

Controllable protein cleavages through intein fragment complementation

Intein‐based protein cleavages, if carried out in a controllable way, can be useful tools of recombinant protein purification, ligation, and cyclization. However, existing methods using contiguous inteins were often complicated by spontaneous cleavages, which could severely reduce the yield of the desired protein product. Here we demonstrate a new method of controllable cleavages without any spontaneous cleavage, using an artificial S1 split‐intein consisting of an 11‐aa N‐intein (I_N) and a 144‐aa C‐intein (I_C). In a C‐cleavage design, the I_C sequence was embedded in a recombinant precursor protein, and the small I_N was used as a synthetic peptide to trigger a cleavage at the C‐terminus of I_C. In an N‐cleavage design, the short I_N sequence was embedded in a recombinant precursor protein, and the separately produced I_C protein was used to catalyze a cleavage at the N‐terminus of I_N. These N‐ and C‐cleavages showed >95% efficiency, and both successfully avoided any spontaneous cleavage during expression and purification of the precursor proteins. The N‐cleavage design also revealed an unexpected and interesting structural flexibility of the I_C protein. These findings significantly expand the effectiveness of intein‐based protein cleavages, and they also reveal important insights of intein structural flexibility and fragment complementation.     

Recombinant oxyntomodulin

An artificial gene encoding oxyntomodulin was obtained using chemical and enzymatic methods and cloned into Escherichia coli. A recombinant plasmid was constructed containing a hybrid oxyntomodulin gene and Ssp dnaB intein from Synechocystis sp. The expression of the resulting hybrid gene in E. coli, its properties, and the conditions of its autocatalytic cleavage to oxyntomodulin were studied.     

Semisynthesis and segmental isotope labeling of the apoE3 N-terminal domain using expressed protein ligation

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that functions as a ligand for members of the LDL receptor family, promoting lipoprotein clearance from the circulation. Productive receptor binding requires that apoE adopt an LDL receptor-active conformation through lipid association, and studies have shown that the 22 kDa N-terminal (NT) domain (residues 1–183) of apoE is both necessary and sufficient for receptor interaction. Using intein-mediated expressed protein ligation (EPL), a semisynthetic apoE3 NT has been generated for use in structure-function studies designed to probe the nature of the lipid-associated conformation of the protein. Circular dichroism spectroscopy of EPL-generated apoE3 NT revealed a secondary structure content similar to wild-type apoE3 NT. Likewise, lipid and LDL receptor binding studies revealed that EPL-generated apoE3 NT is functional. Subsequently, EPL was used to construct an apoE3 NT enriched with ¹⁵N solely and specifically in residues 112–183. ¹H-¹⁵N heteronuclear single quantum correlation spectroscopy experiments revealed that the ligation product is correctly folded in solution, adopting a conformation similar to wild-type apoE3-NT. The results indicate that segmental isotope labeling can be used to define the lipid bound conformation of the receptor binding element of apoE as well as molecular details of its interaction with the LDL receptor.     

蛋白质内含子的特征、转移和演化

自从1990年发现第一个蛋白质内含子,就引起了有关专家的高度重视,蛋白质内含子不仅在理论上丰富了遗传信息翻译后加工的内容,而且实践上在蛋白质纯化方面有着广泛的应用前景.鉴于蛋白质内含子的不断发现,有必要对其在自我剪切,传递,演化等方面的进展做一概述.     

Engineering an affinity tag for genetically encoded cyclic peptides

Peptide expression libraries are valuable probes of cellular function. SICLOPPS technology merges the principal advantages of both genetic methods and small-molecule approaches in yielding superior library sizes of operationally stable, structurally well-defined entities with an established biological and medicinal record. Here, we describe development, application, and the first-generation library implementation of an expressed affinity tag for a library of cyclic peptides. A tripeptide streptavidin-binding motif (HPQ) proved to be compatible with presentation from a backbone cyclized template. A resulting peptide was employed as a sensitive indicator of peptide splicing, expression, and recovery as well as an affinity tag for one-step purification. Specific recognition of the tag by streptavidin was also critical for an analysis of intein mutants. Finally, the initially identified probe was used as a template for design of a streptavidin-responsive cyclic peptide library.     

Characterization of intein homing endonuclease encoded in the DNA polymerase gene of Thermococcus marinus

The DNA polymerase gene of Thermococcus marinus ( Tma ) contains an intein inserted at the pol-b site that possesses a 1611-bp ORF encoding a 537-amino acid residue. The LAGLIDADG motif, often found in site-specific DNA endonucleases, was detected within the amino acid sequence of the intein. The intein endonuclease, denoted as PI- Tma , was purified as a naturally spliced product from the expression of the complete DNA polymerase gene in Escherichia coli . PI- Tma cleaved intein-less DNA sequences, leaving four-base-long, 3'-hydroxyl overhangs with 5'-phosphate. Nonpalindromic recognition sequences 19 bp long were also identified using partially complementary oligonucleotide pair sequences inserted into the plasmid pET-22b(+). Cleavage by PI- Tma was optimal when present in 50 mM glycine–NaOH (pH 10.5), 150 mM KCl and 12 mM MgCl₂ at 70 °C.