Sortase A: A Model for Transpeptidation and Its Biological Applications

2018 ◽  
Vol 34 (1) ◽  
pp. 163-188 ◽  
Author(s):  
Novalia Pishesha ◽  
Jessica R. Ingram ◽  
Hidde L. Ploegh
Keyword(s):  
Directed Evolution ◽  
Living Cells ◽  
Living Systems ◽  
Sortase A ◽  
Biological Applications ◽  
Peptide Bonds ◽  
Protein Modifications ◽  
The Past

Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.

scholarly journals Single-Molecule Kinetics in Living Cells

2019 ◽  
Vol 88 (1) ◽  
pp. 635-659 ◽  
Author(s):  
Johan Elf ◽  
Irmeli Barkefors
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
The Past

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

Toxicity assessment of nanoparticles in various systems and organs

2017 ◽  
Vol 6 (3) ◽  
pp. 279-289 ◽  
Author(s):  
Yuan Yang ◽  
Zhen Qin ◽  
Wei Zeng ◽  
Ting Yang ◽  
Yubin Cao ◽  
...  
Keyword(s):  
Predictive Models ◽  
Toxicity Assessment ◽  
Biological Applications ◽  
Standard Methods ◽  
Immune Systems ◽  
The Past ◽  
Injury Mechanisms ◽  
Histopathological Studies

AbstractIn the past decades, much attention has been paid to toxicity assessment of nanoparticles prior to clinical and biological applications. Whilein vitrostudies have been increasing constantly,in vivostudies of nanoparticles have not established a unified system until now. Predictive models and validated standard methods are imperative. This review summarizes the current progress in approaches assessing nanotoxicity in main systems, including the hepatic and renal, gastrointestinal, pulmonary, cardiovascular, nervous, and immune systems. Histopathological studies and specific functional examinations in each system are elucidated. Related injury mechanisms are also discussed.

Microfluidic Droplet Technique for In Vitro Directed Evolution

2010 ◽  
Vol 63 (9) ◽  
pp. 1313 ◽  
Author(s):  
Nan Wu ◽  
John Oakeshott ◽  
Sue Brown ◽  
Christopher Easton ◽  
Yonggang Zhu
Keyword(s):  
Directed Evolution ◽  
High Throughput Screening ◽  
Rapid Development ◽  
Dna Amplification ◽  
Industrial Applications ◽  
The Past ◽  
Wide Range ◽  
In Vitro Protein Expression ◽  
Mutant Genes

Increasingly over the past two decades, biotechnologists have been exploiting various molecular technologies for high-throughput screening of genes and their protein products to isolate novel functionalities with a wide range of industrial applications. One particular technology now widely used for these purposes involves directed evolution, an artificial form of evolution in which genes and proteins are evolved towards new or improved functions by imposing intense selection pressures on libraries of mutant genes generated by molecular biology techniques and expressed in heterologous systems such as Escherichia coli. Most recently, the rapid development of droplet-based microfluidics has created the potential to dramatically increase the power of directed evolution by increasing the size of the libraries and the throughput of the screening by several orders of magnitude. Here, we review the methods for generating and controlling droplets in microfluidic systems, and their applications in directed evolution. We focus on the methodologies for cell-based assays, in vitro protein expression and DNA amplification, and the prospects for using such platforms for directed evolution in next-generation biotechnologies.

scholarly journals Towards the engineering of in vitro systems

2009 ◽  
Vol 6 (suppl_4) ◽  
Author(s):  
Christoph Hold ◽  
Sven Panke
Keyword(s):  
Rational Design ◽  
Enzymatic Reaction ◽  
Design Procedure ◽  
Reaction Network ◽  
Living Cells ◽  
Living Systems ◽  
Design Parameters ◽  
In Vitro Systems ◽  
Time Invariant

Synthetic biology aims at rationally implementing biological systems from scratch. Given the complexity of living systems and our current lack of understanding of many aspects of living cells, this is a major undertaking. The design of in vitro systems can be considerably easier, because they can consist of fewer constituents, are quasi time invariant, their parameter space can be better accessed and they can be much more easily perturbed and then analysed chemically and mathematically. However, even for simplified in vitro systems, following a comprehensively rational design procedure is still difficult. When looking at a comparatively simple system, such as a medium-sized enzymatic reaction network as it is represented by glycolysis, major issues such as a lack of comprehensive enzyme kinetics and of suitable knowledge on crucial design parameters remain. Nevertheless, in vitro systems are very suitable to overcome these obstacles and therefore well placed to act as a stepping stone to engineering living systems.

How SIMS microscopy can be used in medicine

1992 ◽  
Vol 50 (2) ◽  
pp. 1602-1603
Author(s):  
Philippe Fragu
Keyword(s):  
Mass Spectrometry ◽  
Biomedical Applications ◽  
Secondary Ion Mass Spectrometry ◽  
Chemical Elements ◽  
Biological Applications ◽  
The Past ◽  
Potential Source ◽  
Secondary Ion ◽  
Chemical Integrity ◽  
Scientific Endeavour

The identification, localization and quantification of intracellular chemical elements is an area of scientific endeavour which has not ceased to develop over the past 30 years. Secondary Ion Mass Spectrometry (SIMS) microscopy is widely used for elemental localization problems in geochemistry, metallurgy and electronics. Although the first commercial instruments were available in 1968, biological applications have been gradual as investigators have systematically examined the potential source of artefacts inherent in the method and sought to develop strategies for the analysis of soft biological material with a lateral resolution equivalent to that of the light microscope. In 1992, the prospects offered by this technique are even more encouraging as prototypes of new ion probes appear capable of achieving the ultimate goal, namely the quantitative analysis of micron and submicron regions. The purpose of this review is to underline the requirements for biomedical applications of SIMS microscopy.Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Bioengineered in vitro Vascular Models for Applications in Interventional Radiology

2019 ◽  
Vol 24 (45) ◽  
pp. 5367-5374 ◽  
Author(s):  
Xiaoyun Li ◽  
Seyed M. Moosavi-Basri ◽  
Rahul Sheth ◽  
Xiaoying Wang ◽  
Yu S. Zhang
Keyword(s):  
Interventional Radiology ◽  
Animal Models ◽  
Blood Vessels ◽  
In Vitro Models ◽  
The Past ◽  
Endovascular Interventions ◽  
Conventional Animal ◽  
Vascular Structures

The role of endovascular interventions has progressed rapidly over the past several decades. While animal models have long-served as the mainstay for the advancement of this field, the use of in vitro models has become increasingly widely adopted with recent advances in engineering technologies. Here, we review the strategies, mainly including bioprinting and microfabrication, which allow for fabrication of biomimetic vascular models that will potentially serve to supplement the conventional animal models for convenient investigations of endovascular interventions. Besides normal blood vessels, those in diseased states, such as thrombosis, may also be modeled by integrating cues that simulate the microenvironment of vascular disorders. These novel engineering strategies for the development of biomimetic in vitro vascular structures will possibly enable unconventional means of studying complex endovascular intervention problems that are otherwise hard to address using existing models.

Sortase A Mediated Protein Modifications and Peptide Conjugations

2016 ◽  
Vol 12 (4) ◽  
pp. 205-213 ◽  
Author(s):  
Natalya Voloshchuk ◽  
Danni Liang ◽  
Jun Liang
Keyword(s):  
Sortase A ◽  
Protein Modifications

Novel Findings of Anti-cancer Property of Propofol

2018 ◽  
Vol 18 (2) ◽  
pp. 156-165 ◽  
Author(s):  
Jiaqiang Wang ◽  
Chien-shan Cheng ◽  
Yan Lu ◽  
Xiaowei Ding ◽  
Minmin Zhu ◽  
...  
Keyword(s):  
General Anesthesia ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Intravenous Anesthetic ◽  
The Past ◽  
Anti Cancer ◽  
Underlying Mechanisms ◽  
Recent Attention

Background: Propofol, a widely used intravenous anesthetic agent, is traditionally applied for sedation and general anesthesia. Explanation: Recent attention has been drawn to explore the effect and mechanisms of propofol against cancer progression in vitro and in vivo. Specifically, the proliferation-inhibiting and apoptosis-inducing properties of propofol in cancer have been studied. However, the underlying mechanisms remain unclear. Conclusion: This review focused on the findings within the past ten years and aimed to provide a general overview of propofol's malignance-modulating properties and the potential molecular mechanisms.

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