Overexpressing and Purifying a Toxic Nuclease from Escherichia coli

Summary

This article describes a methodology for overexpressing recombinant Nsp15, a toxic nuclease, in a C41(DE3) expression system, followed by purification of the tagged protein utilizing affinity and size exclusion chromatography. These protocols can be adapted for other challenging toxic proteins.

Abstract

Escherichia coli has been widely employed as an expression system to produce recombinant proteins. Yet some types of recombinant proteins, such as nucleases, can be difficult to overexpress recombinantly in E. coli due to their enzymatic activity on cellular DNA or RNA. Such toxic effects can lead to slow growth and poor protein yields. One such example is the endoribonuclease Nsp15 from coronaviruses: yields of wild-type (WT) gamma-coronavirus Nsp15 are much lower than catalytically inactive Nsp15. To address this issue, we have employed the use of C41(DE3) cells and same-day single colony starter cultures to help reduce toxicity. Recombinant Nsp15 containing an N-terminal 6xHis-tag was overexpressed and purified using cobalt-based affinity chromatography followed by size exclusion chromatography (SEC). The resulting yields of purified protein are sufficient for biochemical assays and cryo-electron microscopy structural studies. This approach has proven effective in mitigating the toxic effects and achieving satisfactory protein yields for further research analysis.

Introduction

Escherichia coli (E. coli) is a bacterium widely used to produce recombinant proteins. Numerous mutant strains, including BL21(DE3) and its derivatives, have been developed to express target proteins¹. The process is typically driven by an encoded bacteriophage T7 RNA polymerase and induced with isopropyl ß-D1-thiopalactopyranoside (IPTG), an analogue of lactose that will activate the lac operon by releasing the lac repressor^1,2,3. Because it is non-hydrolyzable, there is continuous mRNA expression and higher protein pr....

Protocol

1. Preparation for buffer and reagents

1. Overexpression reagents
   1. Prepare 1 L of 2xTY media by combining 16 g of tryptone, 10 g of yeast extract, 5 g of sodium chloride (NaCl), and 900 mL of deionized water in a 2 L baffled flask. Autoclave the flask for at least 30 min on a liquid cycle.
      NOTE: At least 2L of 2xTY media is needed per purification.
   2. Prepare appropriate antibiotic stock/s for the plasmid. For Nsp15-pET-14b, make 15 mL of 100 mg/mL ampicillin by dissolving 1.5 g of ampicillin with deionized water in a 15 mL conical tube. Store the antibiotic stocks at -20 ˚C until needed.
   3. Prepare 250....

Results

Due to Nsp15's toxicity as a nuclease, a total of ~7 h was usually required for the starter cultures to reach an OD₆₀₀ of 0.8-1.0. Once inoculated, a total of ~3-5 h was typically required for the same optimal OD₆₀₀ of 0.8-1.0 for induction. In this case, the doubling time appeared to be ~1 h. When a catalytic-dead mutant of Nsp15 was expressed, the doubling for the E. coli cells appeared normal (~20 min), further evidence that the nuclease activity is responsible for toxicity (

Discussion

We have presented a detailed protocol that results in the successful overexpression in E. coli of a toxic nuclease and purification using affinity and size exclusion chromatography. Implementation of same-day 10 mL single colony starter cultures instead of overnight starter cultures increased consistency in growth times and protein yields across growths. We chose to use C41(DE3) cells, which contain mutations that decrease T7 polymerase activity, to help manage recombinant protein toxicity¹

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm PES, Sterilizing , Low Protein Binding, Filter System, 1 L	Corning	431098
AEBSF serine protease inhibitor	Goldbio	30827-99-7
Agar, bacteriological	Amresco	J637
ÄKTA go purification system	Cytiva	29383015
Amicon Ultra 15 mL, 30 kDa MWCO concentrator	Millipore	UFC903024
Ampicillin sodium	Goldbio	A-301-25
Baffled shake flasks	Nalgene	F0527
Benchtop centrifuge	Eppendorf	5920R
BioPhotometer plus	Eppendorf	E-BP6132
Bovine thrombin	BioPharm	91-030
Bradford dye reagent	Boston BioProducts	BPA-200
Bromophenol blue	Sigma-Aldrich	114931
Calcium Chloride	Sigma-Aldrich	C4901
Digital dry bath	Bio Rad	1660562
Disposable spreaders, L-shaped	MedSupply Partners	62-1014-1
Dithiothreitol	Goldbio	27565-41-9
DNaseI from bovine pancrease	Sigma-Aldrich	D4527
Douce tissue grinder, 40 mL	Wheaton	357546
Econo-column chromagtography columns	Bio-Rad	737-1532
Enduro minimix nutating mixer	Labnet	S0600
Ethanol	Sigma-Aldrich	459844
Excella E25R Inc/Ref Shaker	Eppendorf	NB-E25R
FastPette V2	Labnet	P2000
Fiberlite F10-4x1000 LEX fixed angle rotor	Thermo Fisher Scientific	096-041075
Fiberlite F21-8x50y fixed angle rotor	Thermo Fisher Scientific	096-084275
Gel Doc EZ Imager	Bio-Rad	1708270
Glycerol	Sigma-Alrich	G5516
Glycine	Bio-Rad	161-0718
HEPES	Sigma-Aldrich	H3375
Hot plate stirrer	VWR	12365-346
Hydrochloric acid	VWR	BDH3028
Imidazole	Goldbio	I-902-25
Isopropyl-ß-D-thiogalactioside (IPTG)	Goldbio	12481C25
Lemo21(DE3) competent E. coli cells	New England Biolabs	C2528J
Manganese(II) chloride tetrahydrate	Sigma-Aldrich	M3634
Microcentrifuge tubes, 0.5 mL	VWR	87003-290
Microcentrifuge tubes, 1.7 mL	MedSupply Partners	15-1151
Microcentrifuge tubes, 2 mL	Axygen	MCT-200-C
Mini-PROTEAN tetra vertical electrophoresis cell	Bio-Rad	10025025
Mini-Protean Tris-glycine gels, 4-20%	Bio-Rad	4568094
MyFuge mini centrfuge	MedSupply Partners	C1008-B
Nalgene 1 L super-speed centrifuge bottle	Thermo Fisher Scientific	3141-1006
Nalgene oak ridge centrifuge tube	Thermo Fisher Scientific	31139-0050
Nanophotometer	Implen	3769
Non-shaking incubator	Labnet	I 5110
Overexpress C41(DE3) chemically competent cells	Biosearch Technologies	60442-1
Overexpress C41(DE3) chemically competent cells	Sigma-Aldrich	CMC0017	Discontinued; Biosearch C41(DE3) cells or NEB Lemo21 cells are an alternative
Petri dishes	MedSupply Partners	62-1077-3
Plasmids (pET-14b backbone)	Genscript	Custom order
Plastic micro-cuvettes	Fisher Scientific	14955127
PowerPacuniversal power supply	Bio-Rad	1645070
Precision glide needle	Becton Dixon	305198
Precision plus protein unstained standards	Bio-Rad	1610363
Recovery medium for expression	Sigma-Aldrich	CMR0001K
S-4x1000 swinging bucket rotor	Eppendorf	EP-S4X1
Serological pipette, 10 mL	Faclon	357551
Serological pipette, 25 mL	Faclon	35725
Serological pipette, 5 mL	Faclon	35743
Serological pipette, 50 mL	Pr1ma	PR-Sero-50
Sodium chloride	Sigma-Aldrich	S9888
Sodium dodecyl sulfate	Sigma-Aldrich	L3771
Sodium hydroxide	Sigma-Aldrich	567530
Sonic dismembrator	Fisher Scientific	FB505
Sorvall Lynx 4000 Centrifuge	Thermo Fisher Scientific	75006580
Spephadex G-25 M PD-10 column	Cytiva	17085101
ß-Mercaptoethanol	Millipore	444203
Stainless steele Griffin beaker	Polar Ware	13977-010-EA
Sterile Falcon tubes, 15 mL	Falcon	352097
Sterile Falcon tubes, 50 mL	Falcon	352098
Superdex 200 10/300 GL column	Cytiva	28990944
Syringes, 1 mL	Air-Tite	A1
Syringes, 3 mL	Becton Dixon	309657
TALON metal affinity resin	Takara	635503
Toothpicks	Creativity Street	PAC3690-01
Tris base	Goldbio	T-400-1
Triton X-100	MP Biomedicals	807423
Tryptone	Research Products International	T60065
Vornado	Benchmark	BV101-G
Yeast extract	Research Products International	Y20025