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Related Concept Videos

Operons02:09

Operons

Prokaryotes can control gene expression through operons—DNA sequences consisting of regulatory elements and clustered, functionally related protein-coding genes. Operons use a single promoter sequence to initiate transcription of a gene cluster (i.e., a group of structural genes) into a single mRNA molecule. The terminator sequence ends transcription. An operator sequence, located between the promoter and structural genes, prohibits the operon’s transcriptional activity if bound by a repressor...
Gram-negative Bacterial Protein Secretion Systems01:17

Gram-negative Bacterial Protein Secretion Systems

Gram-negative bacteria utilize sophisticated protein secretion systems to transport proteins across their double-membrane envelope into the extracellular environment or host cells. Based on their mechanism of action, these systems are classified into one-step and two-step pathways.One-Step Secretion Systems (Types I, III, IV, and VI)One-step secretion systems bypass the periplasm entirely, forming a continuous channel that spans both the inner and outer membranes:Type I Secretion System (T1SS):...
Inducible Operons: lac Operon01:25

Inducible Operons: lac Operon

The lac operon in Escherichia coli is a model for understanding inducible gene regulation and metabolic flexibility. It integrates local control by lactose and global regulation through catabolite repression, enabling E. coli to preferentially metabolize glucose when available and switch to lactose utilization when glucose is scarce.Structure and Function of the lac OperonThe lac operon contains three structural genes: lacZ (β-galactosidase), lacY (lactose permease), and lacA (thiogalactoside...
Bacterial Translocation and Protein Secretion01:26

Bacterial Translocation and Protein Secretion

Bacterial protein secretion involves translocation systems to ensure proteins reach their designated locations, including the plasma membrane, periplasm, outer membrane, or the external environment. These translocation systems are vital for bacterial physiology, supporting processes like membrane assembly, enzymatic activity in the periplasm, and interactions with the external environment. The division of labor between Sec and Tat pathways ensures efficiency in handling proteins with diverse...
Formation of Lipopolysaccharides01:19

Formation of Lipopolysaccharides

Lipopolysaccharides (LPS) are crucial components of the outer membrane of Gram-negative bacteria, serving both structural and functional roles. It contributes to membrane stability and protects bacteria from host immune responses. LPS is composed of three major regions—lipid A, a core oligosaccharide, and an O antigen. The biosynthesis and assembly of LPS involve a highly coordinated set of enzymatic reactions and transport mechanisms. Additionally, LPS is recognized as an endotoxin, triggering...
Insertion of Single-pass Transmembrane Proteins in the RER01:26

Insertion of Single-pass Transmembrane Proteins in the RER

Integral membrane proteins are proteins adhered to the lipid bilayer of a cell organelle or membrane. They can be of two types: transmembrane integral proteins that span the lipid bilayer and monotopic proteins that are attached to either side of the membrane but do not pass through it.
Integral transmembrane proteins possess transmembrane and extra membrane domains. The transmembrane domains are primarily made of 20-25 hydrophobic amino acids arranged in a helical secondary confirmation. These...

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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
08:46

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Published on: January 6, 2015

Membrane protein expression in Lactococcus lactis.

Annie Frelet-Barrand1, Sylvain Boutigny, Edmund R S Kunji

  • 1Laboratoire de Physiologie Cellulaire Végétale, CNRS (UMR-5168)/CEA/INRA (UMR-1200), Université Joseph Fourier, iRTSV, CEA-Grenoble, France. annie.barrand-frelet@cea.fr

Methods in Molecular Biology (Clifton, N.J.)
|January 26, 2010
PubMed
Summary
This summary is machine-generated.

This study details a protocol for expressing and detecting membrane proteins using Lactococcus lactis and the nisin-inducible controlled gene expression (NICE) system. It facilitates recombinant membrane protein production with affinity tags for purification.

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Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles
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Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Published on: December 31, 2019

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Membrane proteins are crucial for cellular functions and drug development, yet their structures remain largely uncharacterized.
  • Lactococcus lactis is a versatile host for recombinant protein production, particularly for membrane proteins.
  • The nisin-inducible controlled gene expression (NICE) system offers precise control over heterologous gene expression in L. lactis.

Purpose of the Study:

  • To present a detailed protocol for expressing membrane proteins in Lactococcus lactis.
  • To describe a method for detecting and purifying these proteins using the Strep-tag II affinity tag.

Main Methods:

  • Utilizing the nisin-inducible controlled gene expression (NICE) system in Lactococcus lactis for controlled protein expression.
  • Employing Strep-tag II affinity tags for the detection and purification of recombinant membrane proteins.
  • Extracting and analyzing membrane protein fractions.

Main Results:

  • Successful expression of recombinant membrane proteins in Lactococcus lactis.
  • Efficient detection and purification of membrane proteins using Strep-tag II affinity chromatography.
  • Demonstration of the NICE system's suitability for fine-tuned membrane protein production.

Conclusions:

  • The described protocol provides a robust method for producing and purifying membrane proteins in Lactococcus lactis.
  • This approach enhances the study of membrane protein structure and function.
  • It supports the development of new therapeutic strategies targeting membrane proteins.