重组环状胸腺五肽结构类似物Cyclo-（Cys- Arg-Lys –Asp-Val-Tyr）的制备、鉴定和活性检测

为了实现蛋白内含肽(Intein)介导的重组环状胸腺五肽结构类似物［cyclo-（Cys -Arg-Lys –Asp-Val-Tyr）,cTP］的高效制备,设计并合成编码6个氨基酸的cTP基因,克隆到表达载体pTWIN1,重组表达质粒pTW-cTp转化E.coli ER2566构建工程菌,IPTG诱导由几丁质结合域纯化标签（chitin binding domain,CBD）、2个蛋白内含肽和目的多肽组成的“多元”融合蛋白(CBD-intein1-cTP-intein2-CBD)的高效表达.几丁质柱亲合层析纯化融合蛋白后,改变pH值和温度诱导intein1 C端切割,硫醇MESNA诱导intein2 N端切割,释放N端为Cys,C端为硫酯的重组cTP线性前体,通过非保护多肽硫酯环合法实现环肽生成.激光飞行质谱结果显示,纯化产物的分子量为764.4,与环肽的理论值相符.免疫活性检测结果显示,环肽cTP较线性多肽TP-5具有更显著的促进巨噬细胞吞噬能力的活性（P<0.01）和促进B细胞抗体生成的活性（P<0.01）.     

Protein splicing

Protein splicing is a posttranslational process that results in excision of an internal protein region (intein) and ligation of its flanking sequences (exteins). As distinguished from other variants of protein processing, protein splicing does not require cofactors of enzymes. Protein splicing is catalyzed by an internal domain (so-called Hint domain) of the intein itself. The review considers the main regularities and molecular mechanisms of the process, as well as the functions of Hint domains in other protein families (Hh proteins, bacterial BIL domains, etc.). Studies of protein splicing are of importance from both theoretical and applied viewpoints. For instance, comparisons of the inteins found in different domains of life illustrate the role of horizontal transfer in intein spreading. A possible role of inteins in regulating several cell processes is discussed on the basis of recent data.     

Regulation of protein activity with small-molecule-controlled inteins

Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.     

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.     

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.     

Selection and Structure of Hyperactive Inteins: Peripheral Changes Relayed to the Catalytic Center

Inteins are phylogenetically diverse self-splicing proteins that are of great functional, evolutionary, biotechnological, and medical interest. To address the relationship between intein structure and function, particularly with respect to regulating the splicing reaction, and to groom inteins for application, we developed a phage display system to extend current in vivo selection for enhanced intein function to selection in vitro. We thereby isolated inteins that can function under excursions in temperature, pH, and denaturing environment. Remarkably, most mutations mapped to the surface of the intein, remote from the active site. We chose two mutants with enhanced splicing activity for crystallography, one of which was also subjected to NMR analysis. These studies define a “ripple effect”, whereby mutations in peripheral non-catalytic residues can cause subtle allosteric changes in the active-site environment in a way that facilitates intein activity. Altered salt-bridge formation and chemical shift changes of the mutant inteins provide a molecular rationale for their phenotypes. These fundamental insights will advance the utility of inteins in chemical biology, biotechnology, and medicine.     

High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli

Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.     

Considerations for the process development of insect‐derived antimicrobial peptide production

Antimicrobial peptides (AMPs) could evolve into new therapeutic lead molecules against multi‐resistant bacteria. As insects are a rich source of AMP, the identification and characterization of insect‐derived AMPs is particularly emphasized. One challenge of bringing these molecules into market, e.g., as a drug, is to develop a cost‐efficient large‐scale production process. Due to the fact that a direct AMP isolation from insects is not economical and that chemical synthesis is recommended for peptide sizes below 40 amino acids, a viable option is heterologous AMP production. Therefore, previous knowledge concerning the expression of larger proteins can be adapted, but due to the AMP nature (e.g., small size, bactericide) additional challenges have to be faced during up and downstream processing. Nonetheless the bottleneck for large‐scale AMP production is the same as for proteins; mainly the downstream process. This review introduces opportunities for insect‐derived AMP production, like the choice of the expression system (based on previously derived data), depending on the AMP nature, as well as new purification strategies like elastin‐like peptide/intein based purification strategies. All of these aspects are discussed with regard to large‐scale processes and costs. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:1–11, 2015     

Impacts of the −1 Amino Acid on Yeast Production of Protein-Intein Fusions

Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein allows for site-specific chemical functionalization via expressed protein ligation. It is highly desirable to maximize the yield of functionalizable protein; and previously an evolved intein, 202-08, was identified that could increase protein fusion production in yeast. Given that the −1 amino acid residue upstream of inteins can affect cleavage efficiency, we examined the effects of amino acid variability at this position on 202-08 intein cleavage efficiency and secretion yield. Varying the −1 residue resulted in a wide range of cleavage behaviors with some amino acids yielding substantial autocleaved product that could not be functionalized. Autocleavage was noticeably higher with the 202-08 intein compared with the wild-type Mxe GyrA intein and resulted directly from the catalytic activity of the intein. Refeeding of production cultures with nitrogen base and casamino acids reduced, but did not eliminate autocleavage, while increasing protein-intein production up to seven-fold. Importantly, two amino acids, Gly and Ala, at the −1 position resulted in good cleavage efficiency with no undesirable autocleavage, and can be used in concert with refeeding strategies to increase total functionalizable protein yield for multiple protein fusion partners. Taken together, we describe an optimized yeast expression platform for protein-intein fusions. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2736, 2019     

Ssp DnaB intein大肠杆菌中介导FVIII重链和轻链的连接

研究利用intein的蛋白质反式剪接功能在大肠杆菌中对凝血VIII因子(FVIII)重链和轻链的连接作用,将B结构域大部分缺失型FVIII(BDD-FVIII)于满足剪接所需的保守性氨基酸Ser1657前断裂为重链和轻链,分别与split mini Ssp DnaB intein的106个氨基酸的N端(Int-N)和48个氨基酸的C端(Int-C)融合,构建到原核表达载体pBV220。诱导表达后SDS-PAGE分析可见预期大小的BDD-FVIII蛋白条带,Western blotting用FVIII特异性抗体证明其为剪接所产生的BDD-FVIII蛋白,表明intein可有效连接BDD-FVIII的重链和轻链。为进一步甲型血友病基因治疗研究应用intein以双腺相关病毒载体(AAV)携带FVIII基因,克服单个AAV载体的容量限制提供了依据。