Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

Summary

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Abstract

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)₆-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni²⁺-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Introduction

Prion neurodegenerative diseases, which include bovine spongiform encephalopathy in cattle, scrapie in sheep and Creutzfeldt-Jakob disease in humans, are fatal and incurable. Prion diseases are characterized by conformational changes of cellular prion protein mainly composed of α-helices, into the cross-β-sheet enriched amyloid conformer ^1,2. Cross-β-structures contain densely packed and highly ordered crystalline-like β-sheets stabilized with hydrogen bonds ^3,4. Prion amyloids can self-replicate due to the β-strands at the growing edge that provide a template for recruiting and converting a monomeric protein unit.

Protocol

1. Generation of Plasmid and Shadoo Expression

Note: The gene encoding murine Shadoo protein (Shadoo^25-122), was subcloned into the expression vector pET-28 ¹¹. This clone was transformed into E. coli SoluBL21 bacterial strain, to express Shadoo protein with a N-terminal hexahistidine tag, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG) as explained previously ¹¹. The plasmid can be requested from the authors. The molecular weight of Shadoo calculated from its amino acid sequence is 12,243.2 Da.

Not....

Results

About 5-10 mg of purified Shadoo protein is obtained per liter of bacterial culture. A two-step purification is needed to obtain recombinant Shadoo of high purity. The first step is performed by affinity chromatography with a Ni²⁺-charged column that retains His-tagged-proteins. Eluted fractions are subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (Figure 5A). Fractions containing Shadoo protein are pooled and further purified by size-exclusion chromatography which separates pro.......

Discussion

An easy and efficient protocol for both expression and purification of a large amount of recombinant mouse Shadoo protein is presented. The method described allows successful solubilization and purification of (His₆)-tagged recombinant Shadoo protein. It is important, as pointed out in the title, that when expressed in a prokaryotic bacterium E. coli Shadoo accumulates in inclusion bodies. Prior to Shadoo purification, bacteria have to be lysed by sonication in a buffer containing Triton X-100. Subseq.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AKTA FPLC	GE, Amersham	# 1363
HiLoad 16/60 Superdex	GE Healthcare	17-1069-01
HiTrap IMAC	GE Healthcare	GE17-0920-05
HiPrep26/10 Desalting column	GE Healthcare	GE17-5087-01
SoluBL21 Competent E. coli	Genlantis	C700200
pET-28b+	Novogen	69865-3
IPTG	Sigma-Aldrich	I6758
LB-Broth medium	Sigma-Aldrich	L3022
Eppendorf Biophotometar	Eppendorf	550507804
Trito X-100	Life Technologies	85111
NiSO4	Sigma-Aldrich	656895
EDTA	Sigma-Aldrich	E6758
Tris-HCl	Sigma-Aldrich	RES3098T
Protease Inhibitor Cocktail Tablets	Roche	4693116001
NaCl	Sigma-Aldrich	746398
Imidazol	Sigma-Aldrich	I5513
Gdn-HCl	Sigma-Aldrich	G4505
protein size marker	Thermo Fisher Scientific	26620