异源生物中筛选高剪接活性Intein系统的建立

原始物种体内蛋白质内含子（intein）介导的自催化蛋白剪接反应以100%效率进行.当这些蛋白质内含子被克隆入异源物种时,其剪接效率往往大大降低,绝大多数甚至完全失去剪接能力.本研究根据蛋白质内含子剪接活性与蛋白质外显子（extein）C端第1个保守氨基酸直接相关的特点,设计含有所有这些保守氨基酸的多个短的蛋白质外显子序列,通过PCR引入到卡那霉素抗性蛋白（KanR）的不同位点中,在此外显子中克隆入相应的蛋白质内含子,构建在大肠杆菌中依赖卡那霉素抗性来筛选高剪接活性蛋白质内含子的系统.结果显示,卡那霉素平板上菌落生长的结果与Western印迹检测的结果基本一致.说明建立的筛选高剪接活性蛋白质内含子系统成功.这种含有可选择蛋白质外显子的筛选系统,将蛋白质剪接与卡那霉素抗性相结合,直接从平板上观测剪接结果,成为快速、稳定筛选在异源物种中具有剪接活性蛋白内含子的新手段.     

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.     

大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PA     

重组Maxadilan的制备与鉴定

为利用基因工程技术获得重组Maxadilan(RMMAX), 根据Maxadilan的氨基酸序列, 设计并人工合成了在原核表达的基因。克隆到表达载体pKYB, 重组质粒pKYB-MAX转化表达宿主菌Escherichia coli strain ER2566, 构建表达工程菌。用诱导剂IPTG诱导由目的多肽、内含肽和几丁质结合域(Chitin binding domain, CBD)组成的“三元”融合蛋白表达; 用几丁质珠亲和层析纯化了裂解液中的融合蛋白, 用b-巯基乙醇切割融合蛋白, 获得目的蛋白。所得的多肽经激光飞行质谱测定分子量结果与理论值相符, 生物活性分析表明, 重组Maxadilan有显著的提升血糖的作用。     

Utilization of protein splicing for purification of the human growth hormone

Developing simple and reliable methods to purify recombinant proteins is among the most important problems of modern biotechnology. It is of particular interest to take advantage of protein splicing for this purpose. Affinity tagging of inteins allows the use of regular protocols for protein purification. Autocatalytic excision of the tagged intein yields the pure protein lacking N-terminal formylmethionine. A new purification technique was developed on the basis of protein splicing for the human growth hormone. The Mxe GyrA intein with the histidine tag or cellulose-binding domain was used as a self-removable affinity unit. The resulting two-step purification protocol makes it possible to obtain the human growth hormone having the native N terminus with minimal losses.     

Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides

Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor. Here, a series of precursor recombinants bearing 11 N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMA1-derived homing endonuclease referred to as VDE and as PI-SceI. The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a spliceable precursor model and is then subjected to a 2.1A resolution crystallographic analysis. The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site, and that each of their peptide bonds is in the trans conformation. The S284 O(gamma) atom located at a distance of 3.1A from the G283 C atom in the N-terminal junction suggests that a nucleophilic attack of the C284 S(gamma) atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring. The tetrahedral intermediate is supposedly resolved into a thioester acyl group upon the cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of Nright arrowS acyl shift at the junction between the N-extein and intein. The S738 O(gamma) atom in the C-terminal junction is placed in close proximity to the S284 O(gamma) atom at a distance of 3.6A, and is well suited for another nucleophilic attack on the resultant thioester acyl group that is then subjected to the transesterification in the next step. The reaction steps proposed for the acyl shift are driven entirely by protonation and deprotonation, in which proton ingress and egress is balanced within the splicing site.     

Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein. The X-ray structures indicate that His147 in the conserved block F activates the side-chain N(delta) atom of the intein C-terminal Asn159, leading to a nucleophilic attack on the peptide bond carbonyl carbon atom at the C-terminal splice site. In this process, Arg73 appears to stabilize the transition state by interacting with the carbonyl oxygen atom of the scissile bond. Arg73 also seems to substitute for the conserved penultimate histidine residue in the formation of an oxyanion hole, as previously identified in other inteins. The finding that the precursor structure contains a zinc ion chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2.6 angstroms apart in the free intein structure and 10.6 angstroms apart in the precursor structure. The orientation change of the aromatic ring of Tyr-1 following the initial acyl shift may be a key switching event contributing to the alignment of Arg73 and the C-terminal scissile bond, and may explain the sequential reaction property of the Ssp DnaE intein.     

蛋白质环化的研究进展

天然蛋白质在生物体内主要以线性形式存在,由于多数蛋白质(酶)热稳定性较差,制约了其在工业催化、食品制造、医药领域的高效应用。自然界中发现的天然环肽类物质具有首尾相连的环化结构,使蛋白质具有较高的稳定性,为改造酶的结构、提高其热稳定性及拓宽其应用范围提供了新思路。本文根据国内外在蛋白质环化领域的新动态并结合本实验室的研究,系统介绍了内含肽介导的蛋白质反式剪接、表达蛋白连接、转肽酶催化的转肽作用等几种传统蛋白质环化方法,着重介绍了基于新型超强分子粘合剂Spy Tag/Spy Catcher介导的蛋白质环化的研究。     

Improved protein splicing using embedded split inteins

Naturally split inteins mediate a traceless protein ligation process known as protein trans‐splicing (PTS). Although frequently used in protein engineering applications, the efficiency of PTS can be reduced by the tendency of some split intein fusion constructs to aggregate; a consequence of the fragmented nature of the split intein itself or the polypeptide to which it is fused (the extein). Here, we report a strategy to help address this liability. This involves embedding the split intein within a protein sequence designed to stabilize either the intein fragment itself or the appended extein. We expect this approach to increase the scope of PTS‐based protein engineering efforts.     

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site

Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int^N fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int^N fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int^C fragment of the GOS-TerL intein.