酰氯法合成甘氨酰-甘氨酰-缬氨酸三肽的实验研究

多肽的合成方法很多 ,传统的方法有混合酸酐法、活泼酯法和偶联试剂法 ,本文采用酰氯法 ,以L -缬氨酸为原料 ,经过酰化、氨解形成二肽 ,二肽再经过酰化、氨解最终形成三肽。通过对三肽样品的红外、核磁及高效液相等的鉴定说明该方法实验步骤简单 ,原料易得 ,收率很高 ,很适合于工业化生产 ,目前国内尚无文献报道     

赖氨酸酰化酶的重组表达及其催化合成ε-月桂酰-L-赖氨酸

来源于链霉菌的赖氨酸酰化酶Sm-ELA能催化赖氨酸和月桂酸在水相中合成月桂酰赖氨酸,避免了采用化学合成法所必需的高温和有机溶剂条件,是一种节能、环境友好的替代方法。构建了过表达链霉菌赖氨酸酰化酶基因的重组质粒pET28a-Sm ELA和pTrcOmpXK_(122)Sm ELA,分别实现了该酶在大肠杆菌胞内和细胞表面的活性表达。比较两种不同表达方式的效果后,将重组酶应用于催化合成月桂酰赖氨酸的反应中,结果显示,在赖氨酸浓度为50 mmol/L,月桂酸浓度为10 mmol/L时,反应24 h,月桂酸转化率最高达到31.1%。     

氨酰-tRNA合成酶的研究进展

氨酰-tRNA合成酶催化特异的氨基酸与同源tRNA氨酰化,从而保证了遗传密码翻译的忠实性。这些古老而保守的蛋白质分子除了具有酶的功能外,在哺乳动物细胞中还发现了多种其他功能,具有重要的应用价值。在寻找具有全新作用机制的新抗生素以应对日益严重的抗生素耐药现象过程中,氨酰-tRNA合成酶是细菌蛋白质合成过程中重要的、新颖的靶标,成为关注的重点。定向突变的氨酰-tRNA合成酶可以用来定点掺入非天然氨基酸,扩展蛋白质工程。今后,随着人们对氨酰-tRNA合成酶研究的不断深入,它们还可能用来治疗肿瘤等多种疾病。     

促黄体生成素释放激素的酶促降解

本文着重阐明促黄体生成素释放激素(LH-RH)的酶促降解机制以及影响酶促降解的因素;简要介绍了几种降解酶的物理化学和生物化学性质;并讨论了LH-RH 酶促降解的生理功能。     

赖氨酸酰化酶的重组表达及其催化合成ε-月桂酰-L-赖氨酸

来源于链霉菌的赖氨酸酰化酶Sm-ELA能催化赖氨酸和月桂酸在水相中合成月桂酰赖氨酸,避免了采用化学合成法所必需的高温和有机溶剂条件,是一种节能、环境友好的替代方法。构建了过表达链霉菌赖氨酸酰化酶基因的重组质粒pET28a-SmELA和pTrcOmpXK₁₂₂SmELA,分别实现了该酶在大肠杆菌胞内和细胞表面的活性表达。比较两种不同表达方式的效果后,将重组酶应用于催化合成月桂酰赖氨酸的反应中,结果显示,在赖氨酸浓度为50 mmol/L,月桂酸浓度为10 mmol/L时,反应24 h,月桂酸转化率最高达到31.1%。     

氨酰-tRNA合成酶的进化差异

大量研究显示,细菌与真核生物中的许多氨酰-tRNA合成酶(aaRS)在一些细菌与真核生物中的基因进化机制与模式、氨酰化途径和结构与功能的进化模式等方面往往有着明显的差异。通过对这些差异的深入研究,对于理解蛋白质的结构与功能的进化将是非常有帮助的。虽然造成这些差异的机制目前仍不清楚,但是,所有的这些差异似乎提示,在细菌与真核生物的一些基本生命活动过程中的某些方面,可能还存在着目前尚未被人们所认识到的较大差异。     

大鼠甘氨肽酰化单氨酶基因的克隆与表达

本文采用原位杂交及ＰＣＲ方法,从大鼠脑ｃＤＮＡ库中,筛选到３个大鼠甘氨肽酰化单氧酶基因片段。经ＤＮＡ全序列分析表明,它们跨越ｒＰＡＭ－２全部编码区。通过点突变,ＰＣＲ重组技术等,分别拼接出此双功能酶的ｒＰＨＭ,ｒＰＳＡＬ及其ｒＰＡＭ全构建了多种大肠杆菌表达质粒。     

内吗啡肽-2的人工合成及其酶促降解

内吗啡肽-2(endomorphin-2)是Zadina等人[1]于1997年发现的一种具有镇痛作用的四肽,它存在于动物和人的中枢神经系统内[2].它是内源性μ阿片受体的激动剂,具有高亲合性(K1=690pmol,L)和选择性(δ/μ=13400、K/μ=7600).     

α- 促黑激素的固相合成研究

目的:探索α-促黑激素的合成工艺。方法:采用多肽固相合成法制备α-促黑激素。以Rink amide-MBHA树脂为载体、使用Fmoc保护策略、TBTU、HOBt、DIEA为缩合剂体系,最后用TFA、苯甲硫醚、水、苯酚、乙二硫醇混合液将多肽从树脂上切割下来。结果:合成后的目标多肽产率达64.9%,经过RP-HPLC纯化纯度可达98%,质谱鉴定显示纯化产物与目标多肽理论相对分子质量一致。结论:该方法操作方便,反应结果稳定,为固相合成生产α-促黑激素提供了一种可行的工艺方案。     

Synthesis and Evaluation of Two Coumarin‐Type Derivatization Reagents for Fluorescence Detection of Chiral Amines and Chiral Carboxylic Acids

The synthesis of two fluorescent coumarin‐type chiral derivatization agents ( 4 and 11 ) is reported. A chiral side chain was introduced at position 7 of the coumarin via Mitsunobu reaction. The two coumarins bear in this side chain either a free amino group or a carboxyl group, making them useful for further transformations. Conjugates of chiral prototype drugs with 4 or 11 were prepared by amide coupling of the analyte's carboxyl group to the reagent's amine group, or vice versa. The separation of seven diastereomeric conjugates through achiral high‐performance liquid chromatography (HPLC) on a common C18 column is demonstrated. Chirality 25:957–964, 2013. © 2013 Wiley Periodicals, Inc.     

PTC-氨基酸的HPLC分析

本文综述了氨基酸分析方法的发展,比较了各种方法的优缺点,着重系统地阐述了迅速发展的PTC氨基酸反相HPLC分析新方法的优越性、分析条件和应用。     

非天然氨基酸N-保护的D-(L-)4-吡啶丙氨酸的合成研究

合成了非天然氨基酸N 保护的D (L ) 4 吡啶丙氨酸 ,其结构分别通过旋光度、核磁共振、红外、元素分析得到确证。     

Synthesis and biological characterization of human monocyte chemoattractant protein 1 (MCP-1) and its analogs.

Novel analogs of human monocyte chemoattractant protein 1 (MCP-1) were designed, synthesized and characterized to be used as tools to generate monoclonal antibodies as potential human therapeutics. MCP-1 and three analogs were synthesized by step-wise Fmoc solid phase synthesis. After oxidation to form the two-disulfide bonds, affinity chromatography using an immobilized mouse anti-human MCP-1 monoclonal antibody (mAb) was utilized for a simple and highly effective purification procedure for the proteins. The final products were extensively characterized and compared with recombinant human MCP-1 (rhMCP-1). All proteins showed identical binding with mouse anti-human MCP-1 mAbs as measured by surface plasmon resonance. Synthetic MCP-1 and the analogs were comparable to recombinant MCP-1 in competition radio-ligand binding to CCR2 receptors on THP-1 cells, and MCP-1-induced, calcium mobilization and chemotaxis assays.     

Synthesis of Poly d(G-C) Oligonucleotides

Model sequences for evaluation of the GC dimer sequence repetition on synthesis success were prepared and analyzed by HPLC. Contiguous d(G-C) or d(C-G) sequences have a deleterious effect on DNA oligonucleotide synthesis. The critical number seems to be about 6 GCs in a row. If the GCs are separated by other nucleotides, the effect is not as severe.     

Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays.

Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid-phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach. Fluoride ions were used rather than the conventional organic base piperidine for the repetitive amino-deprotection step. Using a range of acid- and base-labile linkers between the polymer and the peptide, it was shown that free and fully side-chain protected peptides can be obtained using our version of the liquid-phase peptide synthesis method. Protocols for simultaneous multiple syntheses requiring a minimum of equipment are presented and the use of polyethylene glycol-bound peptides in biochemical binding and functional assay systems is demonstrated.     

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5–S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.     

Preparation of 3-Methyl-3-(substituted phenyl)-pyruvic Acid

The relationships between dietary levels of the essential amino acids and hepatic polysome profiles of rats were investigated with special attention to the amino acid requirement pattern for the maximum rat growth as determined by other investigators. The basal diet contained a 7% essential amino acid mixture and a 3% non-essential amino acid mixture, with appropriate amounts of other nutrients. Rats were fed test diet for 5 hours and then the polysome profile was determined. The amounts of essential amino acids needed for maximum aggregation of polysome were low for methionine-cystine, leucine and tryptophan as compared with requirements for maximum growth. But in other essential amino acids, the amounts were in almost the same range as those reported for maximum growth by others. The differences between the amino acid requirement patterns for maximum aggregation of hepatic ribosomes and for maximum growth of rats might be due to a difference in amino acid requirements of the liver and whole body. Therefore, the hepatic polysome profile might be used to measure the effect of amino acid supplementation on dietary proteins. The requirement pattern of essential amino acids in other organs may be studied by polysome profile determination.     

Proteolysis alters the spectral properties of 124 kdalton phytochrome from Avena

Native phytochrome from Avena sativa L. is homogeneous with a monomeric molecular weight of 124 kdalton; 6–10 kdalton larger than the heterogeneous 120 kdalton preparations previously considered to be undegraded (Vierstra and Quail, 1982, Proc. Natl. Acad. Sci. USA, 79: 5272–5276). The phototransformation difference spectrum (Pr-Pfr) of 124 kdalton phytochrome measured in crude extracts has a minimum in the farred region at 730 nm, the same as that observed in vivo. These spectral properties contrast with those of 120 kdalton phytochrome purified by column immunoaffinity chromatography where the difference minimum is at 724 nm. When 124 kdalton phytochrome is incubated as Pr in crude extracts, the difference minimum shifts progressively to shorter wavelengths (from 730 to 722 nm) concomitant with the proteolytic degradation of the chromoprotein to the mixture of 118 and 114 kdalton species that comprise 120 kdalton phytochrome preparations. These two effects are inhibited in concert by the serine protease inhibitor, phenylmethylsulfonylfluoride, and or maintenance of the phytochrome in the Pfr form. These results provide further evidence that 124 kdalton phytochrome is the native molecule in Avena and indicate that the peptide segments removed by proteolysis of the Pr form are important to the pigment's spectral integrity. The present data thus resolve the previously unsettled question of why the Pfr form of 120 kdalton phytochrome isolated by various procedures from Avena has been found to absorb at shorter wavelengths than that observed in vivo. Previous spectral studies with 120 kdalton phytochrome preparations are open to reexamination.Abbreviations, symbols PMSF phenylmethylsulfonylfluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Ig immunoglobulin - A_minimum, A_maximum phototransformation difference spectrum (Pr-Pfr) minimum and maximum - A_r/A_fr ratio spectral change ratio