UID:
almafu_9958132392402883
Umfang:
1 online resource (693 p.)
Ausgabe:
First edition.
ISBN:
0-12-801521-7
Serie:
Methods in Enzymology ; Volume 556
Anmerkung:
Description based upon print version of record.
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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
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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
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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
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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
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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
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5.2. Tips
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English
Weitere Ausg.:
ISBN 0-12-801625-6
Sprache:
Englisch
