降血压肽基因工程菌的构建及高效表达策略

降血压肽具有很好的降血压效果而且天然无毒副作用,是目前基因工程研究的热点之一。本文阐述了降血压肽基因工程菌构建的重要性,综述了国内外关于降血压肽及各种小肽表达方面的研究进展。探讨了降血压肽基因工程菌在构建过程中目的片段的选择、串联连接的方式、串联体的获得及表达载体的选择等方面采用的策略和依据,重点介绍了各种不同表达类型载体的优缺点。旨在能对今后做降血压基因工程菌的多肽表达提供参考,以方便实验设计。     

Peptide-mediated DNA condensation for non-viral gene therapy

The construction of non-viral, virus-like vehicles for gene therapy involves the functionalization of multipartite constructs with nucleic acid-binding, cationic agents. Short basic peptides, alone or as fusion proteins, are appropriate DNA binding and condensing elements, whose incorporation into gene delivery vehicles results in the formation of protein–DNA complexes of appropriate size for cell internalization and intracellular trafficking. We review here the most used cationic peptides for artificial virus construction as well as the recently implemented strategies to control the architecture and biological activities of the resulting nanosized particles.     

Screening of tumor-targeting peptides for diagnosis and therapy through phage display technology

Phage display technology was introduced by G. Smith in 1985, which is highly effective in the selection of affinity peptides from a library containing billions of display peptides. The obtained peptides show potential efficacy in the development of further clinical applications, especially in tumor treatment. In this review, the basic principles, limits, developments of phage display technology and peptide libraries are introduced. Following that, the amino acid sequence of tumor target peptides for hematological and other systems are discussed. Finally, the application of target peptides in the design of imaging probes and the development of target peptide drugs for diagnosis and therapy are noted.     

An improved radioimmunoassay for myelin basic protein-like immunoreactive material in cerebrospinal fluid

A modified radioimmunoassay protocol is described which can measure elevated levels of myelin basic protein-like immunoreactive material in the cerebrospinal fluid of some patients with multiple sclerosis or head injury and in rats developing an acute demyelinating form of experimental allergic encephalomyelitis. The assay uses synthetic peptides that differ in their sequences from natural myelin basic protein for both standard and radioligand, but in apparent contrast to previously published assays serial dilutions of samples produce the expected dose estimates when interpolated from the standard curve. A second radioimmunoassay was produced with high sensitivity and specificity for myelin basic protein peptides with a carboxyl terminus at phenylalanine 89. This assay was used in attempts to detect the myelin basic protein-like immunoreactivity recently reported to occur in human urine. This radioimmunoassay failed to detect specific immunoreactivity in urine samples from control and multiple sclerosis donors. The specificities of both assays were studied using a wide range of synthetic peptides and the importance of this information to the design of future assays is addressed. Our work reinforces that of others in suggesting the complex situation involved in the design of assays for MBP fragments in body fluids. We are willing to distribute the reagents for use in our CSF assay to researchers who request them.     

昆虫抗菌肽和抗真菌肽结构与功能的关系及分子设计

在对GenBank和EMBL数据库中登录的昆虫抗微生物肽 (antimicrobial peptide, AMP), 即昆虫抗菌肽 (antibacterial peptide) 和抗真菌肽 (antifungal peptide, AFP) 进行归类整理的基础上,对天蚕素族(cecropins )抗菌肽结构与功能的关系及人工改造的分子设计策略,特别是对目前新发现的一些昆虫抗真菌肽的已知结构与功能关系的研究进展、存在问题等进行了简要介绍和分析,为从事昆虫抗微生物肽的理论研究和发展新型抗生素药物提供了必要的信息。     

A probabilistic meta-predictor for the MHC class II binding peptides

Several computational methods for the prediction of major histocompatibility complex (MHC) class II binding peptides embodying different strengths and weaknesses have been developed. To provide reliable prediction, it is important to design a system that enables the integration of outcomes from various predictors. The construction of a meta-predictor of this type based on a probabilistic approach is introduced in this paper. The design permits the easy incorporation of results obtained from any number of individual predictors. It is demonstrated that this integrated method outperforms six state-of-the-art individual predictors based on computational studies using MHC class II peptides from 13 HLA alleles and three mouse MHC alleles obtained from the Immune Epitope Database and Analysis Resource. It is concluded that this integrative approach provides a clearly enhanced reliability of prediction. Moreover, this computational framework can be directly extended to MHC class I binding predictions.     

Design of peptides with high affinities for heparin and endothelial cell proteoglycans

Proteoglycan-binding peptides were designed based on consensus sequences in heparin-binding proteins: XBBXBX and XBBBXXBX, where X and B are hydropathic and basic residues, respectively. Initial peptide constructs included (AKKARA)(n) and (ARKKAAKA)(n) (n = 1-6). Affinity coelectrophoresis revealed that low M(r) peptides (600-1,300) had no affinities for low M(r) heparin, but higher M(r) peptides (2,000-3,500) exhibited significant affinities (K(d) congruent with 50-150 nM), which increased with peptide M(r). Affinity was strongest when sequence arrays were contiguous and alanines and arginines occupied hydropathic and basic positions, but inclusion of prolines was disruptive. A peptide including a single consensus sequence of the serglycin proteoglycan core protein bound heparin strongly (K(d) congruent with 200 nM), likely owing to dimerization through cysteine-cysteine linkages. Circular dichroism showed that high affinity heparin-binding peptides converted from a charged coil to an alpha-helix upon heparin addition, whereas weak heparin-binding peptides did not. Higher M(r) peptides exhibited high affinities for total endothelial cell proteoglycans (K(d) congruent with 300 nM), and approximately 4-fold weaker affinities for their free glycosaminoglycan chains. Thus, peptides including concatamers of heparin-binding consensus sequences may exhibit strong affinities for heparin and proteoglycans. Such peptides may be applicable in promoting cell-substratum adhesion or in the design of drugs targeted to proteoglycan-containing cell surfaces and extracellular matrices.     

The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6.

A major challenge in basic and applied biological research is the engineering of small proteins with pre-determined structures and novel functions. In a limited number of cases, this has been achieved by de novo design. An alternative combinatorial approach is based on the construction of large libraries of random peptides and on methods for the selection of the desired molecules. Here we describe a successful combination of both the rational design and the combinatorial approaches for developing proteins with useful biological functions, in this case the construction of a specific inhibitor of the cytokine human interleukin-6. In previous work, the 'minibody', a 61 residue polypeptide consisting of a beta-pleated framework and two hypervariable regions, was designed, synthesized and expressed on f1 phage surface. We report the construction of a repertoire of 50 million minibodies displayed on phage in which the hypervariable regions have been randomized. One polypeptide which binds tightly and specifically to human interleukin-6 was isolated from this collection of minibody mutants. This particular minibody is an effective inhibitor of the cytokine's biological activity. The approach described here could in principle be applied to other molecular targets.     

Foldamers as versatile frameworks for the design and evolution of function

Foldamers are sequence-specific oligomers akin to peptides, proteins and oligonucleotides that fold into well-defined three-dimensional structures. They offer the chemical biologist a broad pallet of building blocks for the construction of molecules that test and extend our understanding of protein folding and function. Foldamers also provide templates for presenting complex arrays of functional groups in virtually unlimited geometrical patterns, thereby presenting attractive opportunities for the design of molecules that bind in a sequence- and structure-specific manner to oligosaccharides, nucleic acids, membranes and proteins. We summarize recent advances and highlight the future applications and challenges of this rapidly expanding field.     

Construction of hybrid peptide synthetases for the production of alpha-l-aspartyl-l-phenylalanine, a precursor for the high-intensity sweetener aspartame.

Microorganisms produce a large number of pharmacologically and biotechnologically important peptides by using nonribosomal peptide synthetases (NRPSs). Due to their modular arrangement and their domain organization NRPSs are particularly suitable for engineering recombinant proteins for the production of novel peptides with interesting properties. In order to compare different strategies of domain assembling and module fusions we focused on the selective construction of a set of peptide synthetases that catalyze the formation of the dipeptide alpha-l-aspartyl-l-phenylalanine (Asp-Phe), the precursor of the high-intensity sweetener alpha-l-aspartyl-l-phenylalanine methyl ester (aspartame). The de novo design of six different Asp-Phe synthetases was achieved by fusion of Asp and Phe activating modules comprising adenylation, peptidyl carrier protein and condensation domains. Product release was ensured by a C-terminally fused thioesterase domains and quantified by HPLC/MS analysis. Significant differences of enzyme activity caused by the fusion strategies were observed. Two forms of the Asp-Phe dipeptide were detected, the expected alpha-Asp-Phe and the by-product beta-Asp-Phe. Dependent on the turnover rates ranging from 0.01-0.7 min-1, the amount of alpha-Asp-Phe was between 75 and 100% of overall product, indicating a direct correlation between the turnover numbers and the ratios of alpha-Asp-Phe to beta-Asp-Phe. Taken together these results provide useful guidelines for the rational construction of hybrid peptide synthetases.     

Allergic encephalomyelitis: characterization of the determinants for delayed type hypersensitivity

Three non-encephalitogenic peptides derived from the encephalitogenic myelin basic protein of the central nervous system, produce delayed type hypersensitivity responses and elicit delayed skin reaction in guinea pigs sensitized with either peptide, the encephalitogenic tryptophan region (peptide E) or the basic protein. The amino acid sequence of the peptides is N-Acetyl-Ala-Ser-Ala-Gln-Lys-OH, forming the N-terminal region of the basic protein molecule, H-Gly-Ser-Leu-Pro-Gln-Lys-OH and H-Gly-Ala-Glu-Gly-Gln-Lys-OH representing residues number 69–74 and 117–122 of the basic protein respectively.     

Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding

Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.     

Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides

Absolute quantification in proteomics usually involves simultaneous determination of representative proteolytic peptides and stable isotope-labeled analogs. The principal limitation to widespread implementation of this approach is the availability of standard signature peptides in accurately known amounts. We report the successful design and construction of an artificial gene encoding a concatenation of tryptic peptides (QCAT protein) from several chick (Gallus gallus) skeletal muscle proteins and features for quantification and purification.     

A multiobjective evolutionary method for the design of peptidic mimotopes.

Peptides that mimic protein epitopes are interesting drug candidates. However, the design of effective peptidic drugs is difficult for several reasons, such as the fast degradation of peptides, their high flexibility, and thus high entropy loss on binding to the target. We therefore propose an in silico method for the automated design of peptides that are optimal with respect to several objectives. We present a Pareto-based multiobjective evolutionary algorithm for in silico peptide design. Using a simple molecular model, we apply the method to the design of peptides that (a) mimic antibody epitopes of the proteins thrombin and blood coagulation factor VIII, respectively, that (b) are short, and (c) are conformationally stable.     

Optimal spatial requirements for the location of basic residues in peptide substrates for the cyclic AMP-dependent protein kinase

The specificity of the cyclic AMP-dependent protein kinase was examined using two series of dodecapeptides as substrates. One series consisted of peptides of the general sequence (Gly)x-Arg-Arg-(Gly)y-Ala-Ser-Leu-Gly in which x + y = 6. The other series consisted of peptides of the sequence (Gly)x-Lys-Arg-(Gly)y-Ala-Ser-Leu-Gly in which x + y was again equal to 6. The peptides Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Arg-Ser-Leu-Gly and Gly-Gly-Gly-Gly-Gly-Gly-Gly-Lys-Arg-Ser-Leu-Gly were also examined. In the series in which the adjacent arginines were located various distances from the serine, the substrate for which the enzyme clearly exhibited optimal kinetic constants contained one amino acid residue between the basic residues and serine. Direct binding studies of N alpha-[3H]acetyl peptides to catalytic subunit of cyclic AMP-dependent protein kinase revealed a correlation between binding affinity and the ability to serve as substrate for the enzyme. In the second series in which the adjacent basic amino acids were Lys-Arg, optimal kinetic constants were again obtained when these residues were separated from serine by a single amino acid. This latter result was surprising in view of phosphorylation site sequences in the known physiologically significant protein substrates for the kinase, since those containing Lys-Arg all contain two amino acids between these residues and serine.     

药学专业《微生物学》实验课程改革

本文对微生物学实验课, 从设计思想、实验模块设计、考核方法、实验教材建设等方面进行了有益的探讨, 尤其是提出在实验课中设置基本技能训练、验证性实验、综合运用性实验、研究性实验等模块; 目的是提高实验课教学效果, 培养具有运用所掌握的理论知识和技能, 创造性地开展科学研究等方面工作能力的良好潜质的人才。     

Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.     

自组装在多肽药物中的应用

自组装是指分子、纳米级结构材料等基本单元自发地组装成一个稳定而又紧密结构的过程。多肽可在各种非共价驱动力下自组装形成纳米纤维、纳米层状结构、胶束等不同的形貌。因多肽具有氨基酸序列明确、易于合成、便于设计等优势,多肽自组装技术成为了近年来的一个研究热点。有研究表明,对某些多肽类药物进行自组装设计或者使用自组装肽材料作为药物递送的载体,可以解决药物自身存在的半衰期短、水溶性差、生理屏障穿透率低等问题。本文重点介绍了自组装多肽的形成机制、自组装形貌、影响因素、自组装设计方法及其在生物医学领域的主要应用,为多肽的高效利用提供参考。     

Experimental allergic encephalomyelitis: basic protein regions responsible for delayed hypersensitivity

Three separate peptide regions were isolated from the chymotrypsin digest of the encephalitogenic basic protein from bovine myelin of the central nervous system. The peptides induced delayed type hypersensitivity (DTH) and elicited delayed skin reactivity in experimental animals. However, none of the isolated peptides was capable of inducing experimental allergic encephalomyelitis (EAE). The amino acid sequence of peptide CTP-3 (Gly-Ala-Glu-Gly-Gln-Lys-Pro-Gly-Phe-OH) and peptide CTP-la were found to overlap the C-terminal sequence of encephalitogenic peptides E (residue 112–125) and T8 (residue 65–74) of the basic protein, respectively. The third DTH inducing peptide, CB1-T1, (N-Acetyl-Ala-Ser-Ala-Gln-Lys-OH) was found to overlap the N-terminal sequence of the basic protein molecule. Common to the three DTH inducing peptides, to the basic protein and to the encephalitogenic peptides E-S and T8S is the X-X-X-Gln-Lys sequence. Isolation of the regions of the basic protein that are responsible for DTH provides antigens for the study of the mechanism of cellular immunity in EAE.