Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Summary

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.

Abstract

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Introduction

Prenyltransferases are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions ^1,2. Z-type enzymes catalyze the formation of cis double bonds from the condensation reaction, whereas E-type enzymes catalyze trans double bond formation ³. cis-Prenyltransferases (cis-PT, Z-type enzymes) are classically classified according to their product chain length into short-chain (C₁₅), medium-chain (C_50-55), and long-chain (C_70-120)

Protocol

1 . Cloning of cis-PT for Overexpression in E. coli

1. Obtain pET-32b expression vector, designed for cloning and high-level expression of protein sequences fused with the 109aa thioredoxin (TRX) protein ¹⁷, and E. coli codon-optimized ¹⁸ coding sequence of full-length cis-PT.
2. Take care to have a TEV-protease (tobacco etch virus protease) cleavage site (ENLYFQ/G, where "/" indicates the cleavage point) ⁹ followed by a short flexible linker (SGSGSG, to enhance cleavage site accessibility) upstream of the cis-PT sequence (

Results

General overview of the construct used here and the purification process are shown in Figure 1. The samples obtained at each purification step are shown in Figure 2. This SDS-PAGE analysis shows the stepwise purification of DHDDS, resulting in a highly purified product. Figure 3 shows the results of analytical SEC of the purified enzyme, revealing that the protein is only observed as a homodimer.

Discussion

The protocol described here for purification of functional human DHDDS in E. coli cells is simple and efficient, allowing one to overexpress and purify the protein in 3 - 4 days once a suitable construct is available. Such protocols for protein purification are of special significance given the breakthroughs in genome sequencing, which provided plethora of information regarding the genetics of many diseases ¹⁸, thereby requiring the development of high-throughput methods to characterize p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pET-32b	Novagen	69016-3
T7 Express lysY Competent E. coli (High Efficiency)	NEB	C3010I
cOmplete, EDTA-free Protease Inhibitor Cocktail	Roche	11873580001
TALON-superflow resin	GE Healthcare	28-9574-99
HiPrep 26/10 desalting column	GE Healthcare	17508701
HiLoad 16/60 superdex-200	GE Healthcare	28989335
superdex-200 increase 5/150 GL	GE Healthcare	28990945
14C-Isopentenyl pyrophosphate	Perkin-Elmer	NEC773050UC
trans,trans-Farnesyl pyrophosphate	Sigma	44270-10MG