Overexpression and Purification of Human <em>Cis</em>-prenyltransferase in <em>Escherichia coli</em>

Ilan Edri; Michal Goldenberg; Michal Lisnyansky; Roi Strulovich; Hadas Newman; Anat Loewenstein; Daniel Khananshvili; Moshe Giladi; Yoni Haitin

doi:10.3791/56430

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

JoVE Journal
Immunology and Infection

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Full Text

7,170 Views

07:35 min

August 3, 2017

DOI: 10.3791/56430-v

Ilan Edri*¹, Michal Goldenberg*¹, Michal Lisnyansky², Roi Strulovich², Hadas Newman^1,3, Anat Loewenstein^1,3, Daniel Khananshvili², Moshe Giladi^2,4, Yoni Haitin²

¹Sackler Faculty of Medicine,Tel Aviv University, ²Department of Physiology and Pharmacology, Sackler Faculty of Medicine,Tel Aviv University, ³Department of Ophthalmology, Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine,Tel Aviv University, ⁴Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine,Tel Aviv University

Overview

This protocol outlines the overexpression and purification of codon-optimized human cis-prenyltransferase under non-denaturing conditions, along with an enzymatic activity assay. It aims to enhance understanding of isoprenoid synthesis and its implications in retinitis pigmentosa.

Key Study Components

Area of Science

Biochemistry
Enzymology
Molecular Biology

Background

Cis-prenyltransferases are crucial for isoprenoid biosynthesis.
Mutations in DHDDS are linked to retinitis pigmentosa.
Understanding these enzymes can provide insights into metabolic pathways.
Current methods for protein production can be inefficient.

Purpose of Study

To develop a simple and effective protocol for protein overexpression.
To purify human DHDDS for further mechanistic studies.
To assess enzymatic activity of the purified protein.

Methods Used

Cloning of human DHDDS gene.
Expression in Escherichia coli.
Purification under non-denaturing conditions.
Enzymatic activity assay to evaluate functionality.

Main Results

Successful overexpression of human cis-prenyltransferase.
High-quality purification achieved under non-denaturing conditions.
Demonstrated enzymatic activity of the purified protein.
Protocol can be adapted for other cis-prenyltransferases.

Conclusions

This method is simple, cost-effective, and time-saving.
It facilitates the study of isoprenoid synthesis mechanisms.
Potential to advance research on retinitis pigmentosa.

Frequently Asked Questions

What is the significance of DHDDS?

DHDDS is involved in isoprenoid biosynthesis and is linked to retinitis pigmentosa.

How does this protocol improve protein production?

It offers a simple and efficient method for overexpressing and purifying proteins.

Can this method be applied to other proteins?

Yes, it can be generalized for other cis-prenyltransferase proteins.

What are the advantages of non-denaturing purification?

It preserves protein functionality and structure, allowing for accurate activity assays.

Is this protocol cost-effective?

Yes, it is designed to be simple and reduce costs associated with protein production.

What type of assays can be performed with the purified protein?

Enzymatic activity assays can be performed to evaluate the functionality of the protein.

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

The overall goal of this protocol is to overexpress and purify codon-optimized human cis-prenyltransferase under non-denaturing conditions and to assay its enzymatic activity. The procedure is demonstrated with the eukaryotic long-chain cis-prenyltransferase dehydrodolichyl diphosphate synthase, or DHDDS. This method can advance our understanding of isoprenoid synthesis and provide key insights into the pathophysiological mechanism of retinitis pigmentosa related to mutations in DHDDS.

The main advantages of this technique is that it's simple, cost-effective, and time-saving. It also can be generalized for high-quantity production of other cis-prenyltransferase proteins. Begin this procedure with cloning an expression of human DHDDS, as described in the text protocol.

View the full transcript and gain access to thousands of scientific videos

Explore More Videos

Cis-prenyltransferase Human DHDDS Overexpression Purification Escherichia Coli IMAC Size Exclusion Chromatography TEV Protease Retinitis Pigmentosa