Membrane proteins-- production and functional characterization. Volume 556

Content: Front Cover
 Membrane Proteins-Production and Functional Characterization
 Copyright
 Contents
 Contributors
 Preface
 Section I: Recombinant Expression of Membrane Proteins
 Chapter 1: Engineering Escherichia coli for Functional Expression of Membrane Proteins
 1. Introduction
 1.1. Green fluorescent protein as a folding reporter
 1.2. Antibiotic resistance marker protein for selection
 2. Preparation of Erythromycin-Sensitive E. coli Strain
 3. Preparation of Expression Plasmid
 3.1. Construction of expression vector for target protein-GFP-ErmC fusion
 3.2. Expression test. 3.2.1. Bacterial culture for protein expression and determination of whole-cell fluorescence3.2.2. Gel-based analysis of the expressed fusion constructs
 4. Selecting Cells for Better Expression
 5. Characterizing Evolved Strains
 5.1. Basic characterizations
 5.1.1. Plasmid copy number, DNA sequencing, and transcript levels
 5.2. Plasmid curing
 5.3. Functional assays
 6. Summary
 Acknowledgments
 References
 Chapter 2: ACEMBLing a Multiprotein Transmembrane Complex: The Functional SecYEG-SecDF-YajC-YidC Holotranslocon Protein S ... 
 1. Introduction
 2. ACEMBLing the HTL Multiprotein Complex. 3. Purifying the HTL4. HTL Integrity and Activity
 4.1. Incorporation of translocation complexes in proteoliposomes
 4.2. Orientation of the reconstituted complexes
 4.3. Subunit interactions and activity of reconstituted translocation complexes
 4.3.1. ATP-stimulated protein secretion by SecA ATPase
 4.3.2. PMF-stimulated protein secretion activity
 4.3.3. Membrane protein insertion activity of the HTL
 5. Discussion and Conclusions
 Acknowledgments
 References
 Chapter 3: Expression and Purification of OsVDAC4
 1. Introduction
 2. Equipment
 3. Materials
 3.1. Solutions and buffers. 4. Protocols4.1. Cloning
 4.2. Transformation protocol for protein expression
 Duration: 2 h + overnight incubation
 4.3. Screening for colonies with high protein expression
 Tips
 4.4. Expression and purification of OsVDAC4
 4.4.1. Soluble OsVDAC4 protein purification
 Duration: 2-3 days
 Tips
 4.4.2. OsVDAC4 purification from inclusion bodies
 Duration: 2 days
 Tips
 4.5. Tryptic digestion of OsVDAC4 for mass spectrometry
 Duration: 2-3 days
 4.6. Functional characterization of OsVDAC4
 4.6.1. Liposome swelling assay
 Duration: 2 days
 Tips
 Note
 4.6.2. Planar bilayer lipid membrane. Duration: 5-6hTips
 Note
 Acknowledgments
 References
 Chapter 4: Membrane Protein Expression in Lactococcus lactis
 1. Introduction
 2. Equipment and Materials
 3. Buffers and Media
 4. Protocol
 4.1. Preparation
 4.2. Duration
 4.3. Tip
 5. Step 1: Cloning the Target Gene into pNZ8048 and Transformation into L. lactis
 5.1. Overview
 Amplification of the Target Gene by PCR
 Restriction digestion
 Preparation of pNZ vector
 Restriction digestion
 Ligation and pellet paint coprecipitation
 Preparation of electrocompetent L. lactis
 Transformation into electrocompetent L. lactis cells.