Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Optimal Foraging00:48

Optimal Foraging

11.9K
How animals obtain and eat their food is called foraging behavior. Foraging can include searching for plants and hunting for prey and depends on the species and environment.
11.9K
Heuristics01:21

Heuristics

58
Heuristics are problem-solving strategies that use mental shortcuts to simplify decision-making. Unlike algorithms, which must be followed precisely to achieve a correct result, heuristics offer a general problem-solving framework. They save time and energy but can sometimes lead to less rational decisions.
People often rely on heuristics when faced with an overload of information, limited time, low importance of the decision, limited information, or when a heuristic readily comes to mind. For...
58
Production Efficiency01:01

Production Efficiency

16.7K
Net production efficiency (NPE) is the efficiency at which organisms assimilate energy into biomass for the next trophic level. Due to low metabolic rates and less energy spent on thermoregulatory processes, the NPE of ectotherms (cold-blooded animals) is 10 times higher than endotherms (warm-blooded animals).
16.7K
The Availability Heuristic01:08

The Availability Heuristic

5.9K
A heuristic is a general problem-solving framework (Tversky & Kahneman, 1974). You can think of these as mental shortcuts that are used to solve problems. Different types of heuristics are used in different types of situations, and the impulse to use a heuristic occurs when one of five conditions is met (Pratkanis, 1989):
5.9K
Maximum Power Transfer01:16

Maximum Power Transfer

193
Numerous practical applications within engineering disciplines, such as telecommunications, necessitate optimizing power delivery to a connected load. This pursuit, however, entails inherent internal losses, which can either equal or exceed the power supplied to the load. The Thevenin equivalent circuit is helpful in finding the maximum power a linear circuit can deliver to a load. It is assumed in this context that the load resistance can be adjusted.
By substituting the entire circuit with...
193
Pharmacokinetic Models: Comparison and Selection Criterion01:26

Pharmacokinetic Models: Comparison and Selection Criterion

31
Physiological and compartmental models are valuable tools used in studying biological systems. These models rely on differential equations to maintain mass balance within the system, ensuring an accurate representation of the dynamic processes at play.
Physiological models take a detailed approach by considering specific molecular processes. They can predict drug distribution, metabolism, and elimination changes, providing a comprehensive understanding of how drugs interact with the body.
31
  1. Home
  3. Research Domains
  5. Engineering
  7. Manufacturing Engineering
  9. Precision Engineering
  11. Optimized Legionella Expression Strain For Affinity Purification Of His-tagged Membrane Proteins Eliminates Major Multimeric Contaminant.
  1. Home
  3. Research Domains
  5. Engineering
  7. Manufacturing Engineering
  9. Precision Engineering
  11. Optimized Legionella Expression Strain For Affinity Purification Of His-tagged Membrane Proteins Eliminates Major Multimeric Contaminant.

Related Experiment Video

A Workflow for Lipid Nanoparticle LNP Formulation Optimization using Designed Mixture-Process Experiments and Self-Validated Ensemble Models SVEM
13:54

A Workflow for Lipid Nanoparticle LNP Formulation Optimization using Designed Mixture-Process Experiments and Self-Validated Ensemble Models SVEM

Published on: August 18, 2023

4.3K

Optimized Legionella expression strain for affinity purification of His-tagged membrane proteins eliminates major multimeric contaminant.

Sukhithasri Vijayrajratnam1, Jonasz B Patkowski2, Joshua Khorsandi3

  • 1Department of Molecular Microbiology, Washington University, St. Louis, Missouri, USA.

Microbiology Spectrum
|May 19, 2025

View abstract on PubMed

Summary
This summary is machine-generated.

We identified and removed a histidine-rich contaminant protein (Lpg1596) that interfered with nickel-nitrilotriacetic acid (Ni-NTA) purification and electron microscopy analysis of Legionella pneumophila. Deleting the lpg1596 gene resolved this issue, enabling cleaner protein isolation.

Keywords:
Legionella pneumophilacryo-EMmembrane proteinspolyhistidine epitope tagged proteins

More Related Videos

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology
06:24

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

Published on: December 15, 2017

9.9K
Author Spotlight: Optimization of Airflow Velocities in Battery Cooling Systems for Enhanced Thermal Performance and Reduced Energy Consumption
10:36

Author Spotlight: Optimization of Airflow Velocities in Battery Cooling Systems for Enhanced Thermal Performance and Reduced Energy Consumption

Published on: November 3, 2023

1.4K

Related Experiment Videos

A Workflow for Lipid Nanoparticle LNP Formulation Optimization using Designed Mixture-Process Experiments and Self-Validated Ensemble Models SVEM
13:54

A Workflow for Lipid Nanoparticle LNP Formulation Optimization using Designed Mixture-Process Experiments and Self-Validated Ensemble Models SVEM

Published on: August 18, 2023

4.3K
Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology
06:24

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

Published on: December 15, 2017

9.9K
Author Spotlight: Optimization of Airflow Velocities in Battery Cooling Systems for Enhanced Thermal Performance and Reduced Energy Consumption
10:36

Author Spotlight: Optimization of Airflow Velocities in Battery Cooling Systems for Enhanced Thermal Performance and Reduced Energy Consumption

Published on: November 3, 2023

1.4K

Area of Science:

  • Microbiology
  • Protein Biochemistry
  • Structural Biology

Background:

  • Polyhistidine-tagged protein purification using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography is common.
  • Contamination by endogenous histidine-rich proteins can compromise purification quality and downstream analysis, such as single particle electron microscopy (EM).
  • An unknown contaminant was observed during the purification of the Legionella pneumophila Dot/Icm type IVB secretion system.

Purpose of the Study:

  • Identify the source of Ni-NTA resin contamination in Legionella pneumophila.
  • Characterize the contaminant protein and its binding mechanism.
  • Develop a strategy to eliminate the contaminant for improved protein purification and analysis.

Main Methods:

  • Mass spectrometry to identify the contaminant protein.
protein purification
single particle analysis
  • Bioinformatic analysis and protein modeling to predict histidine clusters.
  • Construction and analysis of a Legionella pneumophila mutant strain lacking the contaminant gene (∆lpg1596).
  • Ni-NTA affinity purification and negative stain electron microscopy (EM).

    • Main Results:

    • Mass spectrometry identified the contaminant as Lpg1596, a homolog of enoyl-CoA hydratase.
    • Lpg1596 possesses surface-exposed histidine clusters, explaining its affinity for Ni-NTA resin.
    • The ∆lpg1596 mutant strain eliminated Lpg1596 contamination and associated particle formation in Ni-NTA purifications.
    • The ∆lpg1596 mutation did not impair bacterial replication in macrophages, suggesting its utility for pathogenesis studies.

    Conclusions:

    • Lpg1596 is a common Ni-NTA binding contaminant in Legionella pneumophila that interferes with protein purification and EM analysis.
    • Deletion of the lpg1596 gene provides a robust solution for removing this contaminant.
    • The developed ∆lpg1596 mutant strain is suitable for purifying His-tagged proteins from Legionella, facilitating advanced analyses like single particle EM.