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Protein Engineering

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Cover of 'Protein Engineering'

Table of Contents

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    Book Overview
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    Chapter 1 Protein Engineering: Past, Present, and Future
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    Chapter 2 Rational and Semirational Protein Design
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    Chapter 3 Computational Analysis of Protein Tunnels and Channels
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    Chapter 4 YASARA: A Tool to Obtain Structural Guidance in Biocatalytic Investigations
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    Chapter 5 A Computational Library Design Protocol for Rapid Improvement of Protein Stability: FRESCO
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    Chapter 6 Directed Evolution of Proteins Based on Mutational Scanning
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    Chapter 7 A Brief Guide to the High-Throughput Expression of Directed Evolution Libraries
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    Chapter 8 Library Growth and Protein Expression: Optimal and Reproducible Microtiter Plate Expression of Recombinant Enzymes in E. coli Using MTP Shakers
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    Chapter 9 Normalized Screening of Protein Engineering Libraries by Split-GFP Crude Cell Extract Quantification
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    Chapter 10 Functional Analysis of Membrane Proteins Produced by Cell-Free Translation
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    Chapter 11 Practical Considerations Regarding the Choice of the Best High-Throughput Assay
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    Chapter 12 High-Throughput Screening Assays for Lipolytic Enzymes
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    Chapter 13 Continuous High-Throughput Colorimetric Assays for α -Transaminases
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    Chapter 14 Colorimetric High-Throughput Screening Assays for the Directed Evolution of Fungal Laccases
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    Chapter 15 Directed Coevolution of Two Cellulosic Enzymes in Escherichia coli Based on Their Synergistic Reactions
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    Chapter 16 Program-Guided Design of High-Throughput Enzyme Screening Experiments and Automated Data Analysis/Evaluation
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    Chapter 17 Solid-Phase Agar Plate Assay for Screening Amine Transaminases
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    Chapter 18 Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics
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    Chapter 19 Isolation of pH-Sensitive Antibody Fragments by Fluorescence-Activated Cell Sorting and Yeast Surface Display
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    Chapter 20 Library Generation and Auxotrophic Selection Assays in Escherichia coli and Thermus thermophilus
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    Chapter 21 Erratum to: Functional Analysis of Membrane Proteins Produced by Cell-Free Translation
Attention for Chapter 10: Functional Analysis of Membrane Proteins Produced by Cell-Free Translation
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Chapter title
Functional Analysis of Membrane Proteins Produced by Cell-Free Translation
Chapter number 10
Book title
Protein Engineering
Published in
Methods in molecular biology, January 2018
DOI 10.1007/978-1-4939-7366-8_10
Pubmed ID
Book ISBNs
978-1-4939-7364-4, 978-1-4939-7366-8
Authors

Srujan Kumar Dondapati, Doreen A. Wüstenhagen, Stefan Kubick, Dondapati, Srujan Kumar, Wüstenhagen, Doreen A., Kubick, Stefan

Abstract

Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20 μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.

Mendeley readers

The data shown below were compiled from readership statistics for 12 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 12 100%

Demographic breakdown

Readers by professional status Count As %
Researcher 4 33%
Student > Ph. D. Student 4 33%
Professor 1 8%
Student > Master 1 8%
Student > Postgraduate 1 8%
Other 0 0%
Unknown 1 8%
Readers by discipline Count As %
Agricultural and Biological Sciences 6 50%
Biochemistry, Genetics and Molecular Biology 3 25%
Veterinary Science and Veterinary Medicine 1 8%
Unknown 2 17%