Modular co-culture engineering,a new approach for metabolic engineering

With the development of metabolic engineering, employment of a selected microbial host for accommodation of a designed biosynthetic pathway to produce a target compound has achieved tremendous success in the past several decades. Yet, increasing requirements for sophisticated microbial biosynthesis call for establishment and application of more advanced metabolic engineering methodologies. Recently, important progress has been made towards employing more than one engineered microbial strains to constitute synthetic co-cultures and modularizing the biosynthetic labor between the co-culture members in order to improve bioproduction performance. This emerging approach, referred to as modular co-culture engineering in this review, presents a valuable opportunity for expanding the scope of the broad field of metabolic engineering. We highlight representative research accomplishments using this approach, especially those utilizing metabolic engineering tools for microbial co-culture manipulation. Key benefits and major challenges associated with modular co-culture engineering are also presented and discussed.     

A new approach for activation of the kiwifruit cysteine protease for usage in in-vitro testing

Molecular Biology Reports - Actinidin (Act d 1), a highly abundant cysteine protease from kiwifruit, is one of the major contributors to the development of kiwifruit allergy. Many studies have...     

A new approach to tissue engineering of vascularized skeletal muscle

Tissue Engineering of skeletal muscle tissue still remains a major challenge. Every neo-tissue construct of clinically relevant dimensions is highly dependent on an intrinsic vascularisation overcoming the limitations of diffusion conditioned survival. Approaches incorporating the arteriovenous-loop model might bring further advances to the generation of vascularised skeletal muscle tissue. In this study 12 syngeneic rats received transplantaion of carboxy-fluorescine diacetate-succinimidyl ester (CFDA)-labelled, expanded primary myoblasts into a previously vascularised fibrin matrix, containing a microsurgically created AV loop. As control cells were injected into fibrin-matrices without AV-loops. Intra-arterial ink injection followed by explantation was performed 2, 4 and 8 weeks after cell implantation. Specimens were evaluated for CFDA, MyoD and DAPI staining, as well as for mRNA expression of muscle specific genes. Results showed enhanced fibrin resorption in dependence of AV loop presence. Transplanted myoblasts could be detected in the AV loop group even after 8 weeks by CFDA-fluorescence, still showing positive MyoD staining. RT-PCR revealed gene expression of MEF-2 and desmin after 4 weeks on the AVloop side, whereas expression analysis of myogenin and MHC(embryo) was negative. So far myoblast injection in the microsurgical rat AV loop model enhances survival of the cells, keeping their myogenic phenotype, within pre-vascularised fibrin matrices. Probably due to the lack of potent myogenic stimuli and additionally the rapid resorption of the fibrin matrix, no formation of skeletal muscle-like tissue could be observed. Thus further studies focussing on long term stability of the matrix and the incorporation of neural stimuli will be necessary for generation of vascularised skeletal muscle tissue.     

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.     

A new approach for study of structural and functional properties of proteins with unknown functions

Selected proteins were produced in Escherichia coli bacterial expression system--three proteins from extremophil bacteria: a putative monooxygenase from Deinococcus radiodurans, a putative nucleotidyltransferase from Thermotoga maritima, a putative oxidoreductase from Exiguobacterium sibiricum; and a shaperon from Homo sapiens DJ-1. The protocol of isolation & purification of recombinant proteins were developed that allowed to obtain expression products with the purity of no less than 96%. Conditions for the crystallization have been selected that allowed a stable growth of crystals. Preliminary x-ray experiments were conducted in order to confirm the quality of produced crystals; the resolution of obtained structural data was from 1.2 to 1.8 angstrom.     

A new method for protease activity measurement.

A new method for protease activity measurement is described. In the presence of excess leucine aminopeptidase from Aspergillus japonica, action of protease on succinyl-casein results in the production of l-amino acids and their amino acids are simultaneously determined by l-amino acid oxidase-peroxidase system. Our proposed method is less time consuming and has a much higher sensitivity than the casein-Folin method. The present method is suggested to be suitable for the assay of neutral or alkaline proteases from animals and microorganisms.     

Patch engineering: a general approach for creating proteins that have new binding activities

Patch engineering is a technique for creating folded proteins that have new binding activities. Different protein scaffolds are used to present a patch of discontinuous residues on a folded-protein surface. By varying simultaneously the residues in these patches and displaying these mutant proteins on phage, one can select proteins that have new binding activities. Patch engineering is applicable to any protein fold. Novel proteins derived by this approach might replace antibodies in certain applications or provide lead molecules for the design of non-peptide analogues.     

Isolation of subtilisin pro-sequence mutations that affect formation of active protease by localized random polymerase chain reaction mutagenesis

In order to analyze the role of the pro-sequence in folding of the alkaline serine protease subtilisin, localized random mutagenesis using the polymerase chain reaction with Taq DNA polymerase was employed to obtain mutations in the pro-sequence which prevent production of active protease. The unique aspect of this procedure is that random mutations can be easily generated in vitro over large but defined regions of a specific gene. The method was applied to a 458-base pair fragment encompassing the coding region of the pro-sequence of subtilisin, a region of the protein which has been shown to be required for proper folding. Protease-deficient mutants containing a variety of amino acid substitutions were isolated with a frequency of 4.3%. From analysis of these mutants, four independent amino acid substitution mutations in the pro-sequence were identified. The present results demonstrate that polymerase chain reaction is an efficient and simple method for obtaining random mutations within a localized region of a given gene.     

A traveling salesman approach for predicting protein functions

Background  

Protein-protein interaction information can be used to predict unknown protein functions and to help study biological pathways.     

The role of the pro-sequence in the processing and secretion of the thermolysin-like neutral protease from Bacillus cereus

The Bacillus cereus cnp gene coding for the thermolysin-like neutral protease (TNP) has been cloned, sequenced, and expressed in Bacillus subtilis. The protease is first produced as a pre-pro-protein (M(r) = 61,000); the pro-peptide is approximately two-thirds of the size of the mature protein. The pro-sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro-sequences are shown to be homologous to the pro-sequence of Pseudomonas aeruginosa elastase. A mutant has been constructed from cnp, in which 23 amino acids upstream from the pro-protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro-sequences. The deletion results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production. N-terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro-sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secretion of the pro-protein.     

生态系统途径——生态系统管理的一种新理念

介绍了生态系统途径的概念和内涵．生态系统途径最早由西方生态学家提出。随后得到一系列国际学术组织和国家的认同和支持,其中《生物多样性公约》、世界自然保护联盟和世界野生动物基金发挥了重要作用,．生态系统途径的实质是对土地、水和生物资源进行综合管理,旨在生态系统保护、生物资源可持续利用和共享生物资源三者之间达到平衡．作为生态系统管理的一种方法论,它以生物为核心。将人类及文化的多样性视为生态系统的一个组成部分,2000年《生物多样性公约》缔约国会议上制定的生态系统管理的12条基本原则和5项行动指南,丰富了生态系统途径的内涵,明确了实施的办法,我国在生态系统管理方面有着丰富的学术储备和经验总结。但也存在一定问题。

     

干细胞研究的新途径:miRNAs

微小RNA(microRNAs,miRNAs)是一类内源性的非编码单链RNA,能够通过与靶mRNA特异性的碱基配对而导致靶mRNA降解或抑制其翻译,从而对基因进行转录后调控。干细胞的自我更新和多向分化过程依赖于广泛而多样的调控机制,miRNAs正是这些调控机制中非常重要的一类分子。研究发现,干细胞的自我更新功能需要多种miRNAs的参与来维持;干细胞的分化也是多种miRNAs参与调控的结果。miRNAs可以作为干细胞研究的一个新的切入点。     

ER-mediated phagocytosis: a new membrane for new functions

Genomics and other high-throughput approaches, such as proteomics, are changing the way we study complex biological systems. In the past few years, these approaches have contributed markedly to improving our understanding of phagocytosis. Indeed, the ability to study biological systems by monitoring hundreds of proteins provides a level of resolution that is not attainable by the usual 'one protein at a time' approach. In this article, I discuss how proteomic approaches have revealed surprising findings that enable us to revisit established concepts, such as the origin of the phagosome membrane, and to propose new models of cell organization and the link between innate and adaptive immunity.     

A dual protease approach for expression and affinity purification of recombinant proteins

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His₆-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His₆-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His₆ tag is removed by His₆-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His₆-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification.