Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Summary

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

Abstract

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Introduction

Clostridium difficile is one of the major causes of nosocomial antibiotic-associated diarrhea infections¹. This Gram-positive anaerobic bacterium is transmitted through its spore form via the fecal-oral route. In the past decade, new ''epidemic'' or ''hypervirulent'' strains (e.g. BI/NAP1/027) caused a drastic increase in new infections and fatality rates in North America and Europe². C. difficile-associated disease (CDAD) is a life threatening colon inflammation with high fatality rates³. The symptoms range from diarrhea⁴ to pseudomembranous colitis⁵ and ....

Protocol

1. Cloning and Construct Design

1. Clone the codon-optimized sequence (for E. coli) of C. difficile PPEP-1 without the signal peptide [amino acids 27-220, named hereafter recombinant PPEP-1 (rPPEP-1)¹¹] into the pET28a vector using NdeI and XhoI restriction sites (Figure 1) with a stop codon at the 3'-end (resulting vector pET28a-NHis-rPPEP-1). This produces a N-terminally 6xHis-tagged protein (NHis-rPPEP-1) with a thrombin cleavage site allowing removing the tag during purification (Figure 1). The plasmid contains a kanamycin resistance cassette for selection. The primers used f....

Results

rPPEP-1 is overexpressed in several E. coli strains, with the highest yield in E. coli BL21 (DE3) Star (Figure 1C). After the first NiNTA affinity chromatography step the 6xHis-tag can be successfully cleaved off from most of the protein and in the second NiNTA step undigested protein can be completely separated from thrombin-digested protein (Figure 1D). On a S200 16/600 column untagged rPPEP-1 migrates as monomer with occasional fronti.......

Discussion

X-ray crystallography is still the fastest and most accurate method to determine three-dimensional near-atomic resolution structures of proteins²⁸. However, it requires the growth of well-ordered single crystals. These are often difficult to get and the crystalline state is artificial. However, a comparison of protein structures determined by X-ray crystallography with those determined by other methods, especially NMR, shows generally a very good agreement. In the case of PPEP-1, an NMR structure published rec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Genes / Vectors / cell strains
pET28a vector	Merck-Millipore	69864	Thrombin cleavable N-terminal His-tag
E. coli strain BL21 (DE3) Star	ThermoFisher Scientific	C601003	RNase H deficient
Codon-optimized gene (for E. coli) of PPEP-1 (CD630_28300)	Geneart (Thermo Fisher Scientific)	custom	amino acids 27-220
Name	Company	Catalog Number	Comments
Chemicals
Yeast extract	any
Tryptone	any
Antifoam B	Sigma-Aldrich	A5757	aqueous-silicone emulsion
Agar	any
Kanamycin	any
IPTG	AppliChem	A1008
Tris-HCl	AppliChem	A1087	Buffer grade
NaCl	any		Buffer grade
DNaseI	AppliChem	A3778
Imidazole	AppliChem	A1073	Buffer grade
Thrombin	Sigma-Aldrich	T4648
Ammonium phosphate dibasic	Sigma-Aldrich	215996
Glycerol 100%	any		purest grade
Sucrose	Sigma-Aldrich	84097
Liquid nitrogen	any		for storage and cryocooling of crystals
Name	Company	Catalog Number	Comments
Equipment (general)
Shaking incubator	any		providing temperatures of 20 °C - 37 °C
Glassware	any		baffled Erlenmeyer flasks (50 ml - 2.8 L)
Centrifuge for large culture volumes	any		centrifuge for processing volumes up to 12 L
Sonicator Vibra-Cell VCX500	Sonics	SO-VCX500	or any other sonicator / cell disruptor
Ultracentrifuge	any		centrifuge providing speeds up to 150,000 x g
NiNTA Superflow resin	Qiagen
Empty Glass Econo-Column	Bio-Rad	7371007	or any other empty glass or plastic column
Size exclusion chromatography column HiLoad Superdex 200 16/600	GE Healthcare	28989335
Chromatography system Äkta Purifier	GE Healthcare	28406264	or any other chromatography system
Dialysis tubing Spectra/Por 3	Spectrum Labs	132724
Dialysis tubing closures	Spectrum Labs	132738
Ultrafiltration units (concentrators) 10,000 NWCO	any
UV-Vis spectrophotometer	any
Name	Company	Catalog Number	Comments
Equipment (crystallography)
Low volume pipette 0.1-10 µl	any
Positive displacement pipette Microman M10	Gilson	F148501
Crystallization robot	any
96-well crystallization plates TTP IQ with three protein wells	TTP	4150-05810	or any other 96-well crystallization plate
24-well CombiClover Junior Plate	Jena Bioscience	EB-CJR
Crystal Clear Sealing Tape	Hampton Research	HR3-511
Siliconized Glass Cover Slides	Hampton Research	HR3-225
Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal	Hampton Research	diverse
Commercial crystallization screens: Wizard, PACT++, JCSG++	Jena Bioscience	diverse
JBS Beads-for-Seeds	Jena Bioscience	CO-501
CrystalCap SPINE HT (nylon loops)	Hampton Research	diverse	loop sizes 0.025 mm - 0.5 mm
CrystalCap Vial	Hampton Research	HR4-904
Cryogenic Foam Dewar 800 ml	Hampton Research	HR4-673
Cryogenic Foam Dewar 2 L	Hampton Research	HR4-675
Vial Clamp, Straight	Hampton Research	HR4-670
CrystalWand Magnetic, Straight	Hampton Research	HR4-729
CryoCane 6 Vial Holder	Hampton Research	HR4-711
CryoSleeve	Hampton Research	HR4-708
CryoCane Color Coder - White	Hampton Research	HR4-713
Scalpel	any
Straight microforcep	any		for manipulation of sealing tape. etc.
Acupuncture needle	any		e.g. from a pharmacy
Stereo microscope	any		for inspection of crystallization plates and crystal mounting, magnification up to 160X