The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Summary

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

Abstract

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3’-5’ exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-β-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

Introduction

The introduction of overexpression technologies boosted either the biochemical studies of low-copy number enzymes and the industrial production of pharmacologically active proteins (e.g., insulin). Since the advent of these technologies, significant advances have been achieved to increase the yield and quality of recombinant proteins. In addition, prokaryotic^1,2 and eukaryotic³ overexpression systems were developed over the years, offering useful alternatives to the “work horse” of protein biotechnology, i.e., Escherichia coli. In particular, the availability of alternative platforms to E. coli led to the production of recombinant peptides or proteins bearing post-translational modifications. However, it should be mentioned that E. coli still represents the organism of choice for recombinant protein production. This is due to several factors, among which the most relevant can be considered: i) the availability of quite a number of overexpression systems (expression vectors and strains) for E. coli^1,2; ii) the short generation times, and high biomass yields, of E. coli in a variety of rich and synthetic media; iii) the facile manipulation either at the biochemical and at the genetic level of this microorganism; iv) the isolation of strains capable of the production of toxic proteins⁴; v) the construction of strains featuring homogeneous induction at the population level^5,6. In addition, it was recently shown that expression systems suitable for the production in E. coli of post-translationally modified proteins can be devised and constructed².

At present, protein overexpression is mainly used to obtain monomeric or homo-oligomeric proteins, whose hypersynthesis can be performed with a single gene cloned into an appropriate plasmid. However, attention was recently paid to the construction of E. coli protein co-expression systems, challenging the production, in vivo, of hetero-oligomeric complexes². Interestingly, early experiments of protein co-expression addressed the inter-species assembly of large and small subunits of cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase^7,8, and the association of truncated and full-length forms of HIV-1 reverse transcriptase⁹. These pioneering studies demonstrated that protein co-expression represents a powerful alternative to traditional in vitro reconstitution. In addition, protein co-expression in E. coli was used to produce different proteins bearing post-translational modifications², to obtain proteins containing unnatural amino acids², and to increase the yield of overexpressed membrane proteins². Moreover, the potential of protein co-expression as a tool to confer to E. coli competence in protein secretion is under active investigation².

Two main strategies of protein co-expression in E. coli can be pursued: i) the use of a single plasmid to host the different genes to be overexpressed; ii) the use of multiple plasmids in single cells to co-express the target proteins. In the first case, the criteria for the choice of plasmid do not differ from those of traditional single protein overexpression experiments, although particular plasmids containing tandem promoter/operator elements were constructed for co-expression¹⁰. This first approach is therefore quite simple. However, it should be mentioned that the use of a single plasmid to co-express different proteins faces two major difficulties: i) the molecular mass of the vector increases with the number of hosted genes, limiting the number of co-expressed proteins; ii) when multiple genes are cloned under the control of a single promoter, polarity can decrease the expression of the genes distal from the promoter. The use of dual or multiple plasmids in single E. coli cells has to accomplish the compatibility of the vectors of choice, therefore imposing constraints to the eligible combinations of plasmids. However, this second co-expression strategy features the advantage of containing the molecular mass of vectors, and limits polarity. We recently constructed a protein co-expression system designed to facilitate the shuttling of genes between the co-expression plasmids¹¹. In particular, we constructed the pGOOD vectors series, the relevant features of which are: i) a p15A origin of replication, to provide compatibility of the pGOOD plasmids with the commercial vectors containing the ColE1 origin (e.g., the pBAD series¹²); ii) a tetracycline-resistance cassette; iii) the presence of lac-derived regulatory elements, i.e., the Promoter-Operator(O₁) couple and the lacI^q gene. Using an appropriate pBAD-pGOOD couple, we were able to overexpress the catalytic core of E. coli DNA polymerase III, composed of three different subunits, i.e., α (the 5’-3’ polymerase), ε (the 3’-5’ exonuclease) and θ (stabilizing ε)¹³. In particular, we demonstrated that the co-expression of the αεθ complex was strictly dependent on the addition to the E. coli culture medium of both IPTG and arabinose, triggering overexpression from pGOOD and pBAD, respectively (Figure 1A).

In the present report, we illustrate how protein co-expression can efficiently solve difficulties linked to the poor solubility of a protein complex subunit. In addition, we show how in vivo protein complementation tests can be performed, and we finally report on the use of protein co-expression to tune mutation frequency in E. coli. To this aim, we used pGOOD-pBAD couples suitable to illustrate relevant examples of each case study.

Protocol

1. Isolation of E. coli Co-transformants

1. Prepare electro-competent cells of the appropriate E. coli strain to be transformed. Transfer to 1 ml of LB medium (Tryptone, Yeast Extract, NaCl at 10, 5, and 10 g/L, respectively) a single colony of the strain of choice, and incubate at 37 °C under shaking conditions of 180 rpm. Dilute the pre-culture 1:500 in 25 ml of fresh LB medium and incubate the culture at 37 °C.
2. At log-phase (0.6 O.D.) centrifuge the cells suspension at 5,000 x g for 20 min), and resuspend the pellet in 10%, v/v ice-cold sterile glycerol-water in half of the original culture volume. Repeat this step 4 times, halving each time the resuspension volume. Finally, resuspend the pellet in glycerol/water and divide the cells suspension in 50 μl aliquots. Store the aliquots at -80 °C up to 6 months.
3. Dissolve the desired plasmid in sterile water supplemented with 0.5 mM EDTA. Thaw on ice an aliquot of electro-competent cells and mix with an appropriate amount (2.5-5 ng) of vector. Dispense the mixture into a 0.1 cm cuvette suitable for electroporation, and apply 1.8 kV.
4. Immediately transfer the electroporated cells into 1 ml of SOC medium (LB medium supplemented with 0.2% w/v glucose, 10 mM MgCl₂, 2.5 mM KCl), incubate for 1 hr under shaking, and finally transfer 100 μl aliquots to Petri dishes containing LB agar with the appropriate antibiotic. Incubate O/N at 37 °C.
5. Purify the transformants by streaking single colonies on Petri dishes.
6. Prepare electro-competent cells of the primary transformants and repeat steps 1.1 to 1.5 to transform with further plasmids.
7. Prepare glycerol stocks of the co-transformants. Transfer a single colony in LB supplemented with the appropriate antibiotics, incubate at 37 °C under shaking, and at log-phase (0.6 O.D.) centrifuge the cells suspension at 5,000 x g for 20 min. Resuspend the pellet in LB-antibiotics medium containing 15% v/v glycerol. Dispense in aliquots and store at – 80 °C.

2. Co-expression of α and ε Subunits of E. coli DNA Polymerase III

1. Transfer with a sterile loop a small amount of the appropriate strain glycerol stock (e.g., TOP10/pGOOD-ε243 and TOP10/pGOOD-ε243-θ/pBADα1160, see Figure 1) into a Petri dish containing LB medium and antibiotics. Let the cells suspension dry onto the Petri dish, and streak the cells droplet. Incubate at 37 °C O/N.
2. Transfer, using a sterile toothpick, a single colony to 1 ml of LB-antibiotics medium. Incubate 8 hr at 37 °C. Dilute the pre-culture 1:500 in fresh medium and incubate at 30 °C for 15 hr.
3. Add IPTG, arabinose, or arabinose and IPTG, 1 mM each. Incubate at 30 °C for 2.5 hr. Collect the cells, and store the pellets at -20 °C.
4. Thaw pellets on ice and resuspend in lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 2.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8). Gently homogenize the cells suspension with a cold glass potter.
5. Sonicate at 15 W for 15 sec, followed by 15 sec cooling interval (4 cycles).
6. Centrifuge the total protein extracts at 10,000 x g for 20 min, at 4 °C to recover the soluble fraction.
7. Analyze by SDS-PAGE (12.5% acrylamide) aliquots of each soluble protein extract. To this aim, transfer 20 μl of each soluble protein extract to Eppendorf tubes containing 60 μl of H₂O and 20 μl of loading buffer (250 mM Tris-HCl pH 6.8, 10 mM β-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol, 0.25% (w/v) bromophenol blue) and boil for 5 min. Load 18 μl of each sample and perform the electrophoresis at 140 V for about 1.5 hr.

3. Co-expression of α Subunit of Deinococcus radiodurans DNA Polymerase III and ε Subunit of E. coli DNA Polymerase III

1. Prepare a pre-culture of the E. coli strain TOP10/pBAD-αDr/pGOOD-ε243 in 1 ml of LB-ampicillin-tetracycline medium and incubate 8 hr at 37 °C. Dilute 1:1,000 in fresh medium and incubate O/N at 37 °C.
2. Dilute 1:100 into 200 ml of fresh medium, incubate at 37 °C for 3 hr, then induce for 3 hr with arabinose and IPTG, 1 mM each. Harvest the cells, and store the pellet at -20 °C.
3. Resuspend the pellet in 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF. Homogenize and disrupt the cells as described in 2.4 and 2.5.
4. Immediately centrifuge the cells lysate at 10,000 x g for 20 min. Discard the pellet. Filter the supernatant with a Büchner funnel equipped with 3 layers of paper filter. Apply gentle vacuum. To avoid excessive foaming, keep the vacuum Erlenmeyer flask on ice during filtration.
5. Determine protein concentration according to Bradford¹⁴.

4. Gel Filtration Chromatography

1. Equilibrate a water-jacketed 16x70 (200) gel filtration column with 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8. Load onto the column the soluble protein extract. For optimal resolution, use a 1 ml sample loop. Perform the chromatography at 0.6 ml/min. Keep column temperature at 4 °C throughout.
2. Collect 0.9 ml fractions and analyze them by SDS-PAGE. Transfer 20 μl of each relevant fraction to Eppendorf tubes containing 60 μl of H₂O and 20 μl of loading buffer (250 mM Tris-HCl pH 6.8, 10 mM β-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol, 0.25% (w/v) bromophenol blue) and boil for 5 min. Load 18 μl of each sample and perform the electrophoresis at 140 V for about 1.5 hr.
3. Determine the 3’-5’ exonuclease and the DNA polymerase activity of each fraction in 96-well microplates. Assay the exonuclease activity using thymidine 5’-monophosphate p-nitrophenyl ester (pNP-TMP) as substrate¹⁵. Estimate DNA polymerase activity using the PPX (Pyrophosphatase, Purine nucleoside phosphorylase, Xanthine oxidase) enzyme-coupled assay¹⁶ and 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) as the electron acceptor¹⁶.

5. Mutation Analysis of Populations Co-expressing the Wild-type ε Subunit of E. coli DNA Polymerase III and the Mutagenic εD12A Variant

1. Transfer a single colony of E. coli TOP10 containing the pBAD-ε and the pGOOD1-εD12A¹¹ vectors to 1 ml of LB-antibiotics medium. Incubate O/N at 37 °C.
2. Dilute the pre-culture 1:250 in 3 flasks containing fresh medium (10 ml), add the appropriate inducers (arabinose, IPTG, or arabinose and IPTG, 1 mM each) and incubate at 37 °C for 8 hr. Prepare in parallel non-induced cultures.
3. Collect aliquots of 1 ml, then dilute 1:500 in new flasks containing fresh medium (10 ml) supplemented or not with the appropriate inducers. Incubate O/N at 37 °C.
4. Repeat steps 5.2 and 5.3 and collect aliquots of 1 ml.
5. Determine the number of generations occurred in each culture. Transfer on LB plates, 100 μl of appropriate serial dilutions of inoculum and culture. Incubate O/N at 37 °C. Count the colonies on the LB plates, and calculate the log of the number of cells present in the inoculum (log_I) and in the culture at the end of growth (log_C). To determine the number of generations, use the formula: (log_C– log_I)/0.301.
6. Centrifuge at 5,000 x g for 20 min and resuspend the cells in 1 ml of 50 mM Tris-HCl pH 7.6, 50 mM NaCl. To permeabilize the cells, add 2-3 drops of chloroform and vortex for 20 sec.
7. Determine the β-glucosidase activity of each aliquot in a 96-well microplate, using p-nitrophenyl-β-D-glucopyranoside (PNPGluc) as substrate. Add to each well 100 µl of permeabilized cells and 100 µl of substrate (16 mg/ml stock solution in H₂O). Take care to avoid air bubbles in the wells. Read the Absorbance at 420 nm, using a microplate reader and the appropriate filter.

Results

The ε subunit of E. coli DNA polymerase III consists of 243 amino acids and features poor solubility^17,18, unless the residues 187-243 are deleted¹⁷. However, we have previously shown¹¹ that the co-expression of full-length α, ε, and θ subunits yields soluble DNA polymerase III catalytic core (Figure 1). In particular, using the pBAD-pGOOD co-expression system, we demonstrated that: i) the overexpression of α and ε subunits can be in...

Discussion

Proteins can be intrinsically disordered, featuring regions whose tertiary structure is not restricted to a limited number of conformations²⁵. These disordered proteins are usually prone to aggregation²⁵, and their isolation and characterization might represent a difficult task. The ε subunit of E. coli DNA polymerase III features two distinct domains^26,27, namely: i) the N-ter domain, bearing the 3’-5’ exonuclease activity, and competent in binding the θ su...

Materials

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
Agar	Sigma-Aldrich	A1296
Ampicillin	Sigma-Aldrich	A9518
Chloroform	Sigma-Aldrich	288306
EDTA	Sigma-Aldrich	EDS
Glycerol	Sigma-Aldrich	G5516
INT	Sigma-Aldrich	I8377
IPTG	Sigma-Aldrich	I5502
KCl	Sigma-Aldrich	P9541
L-Arabinose	Sigma-Aldrich	A3256
MgCl2	Sigma-Aldrich	M2670
NaCl	Sigma-Aldrich	31434
PMSF	Sigma-Aldrich	P7626
PNP-gluc	Sigma-Aldrich	N7006
pNP-TMP	Sigma-Aldrich	T0251
Tetracyclin	Sigma-Aldrich	87128
Trizma base	Sigma-Aldrich	T1503
Tryptone	Sigma-Aldrich	95039
Yeast extract	Fluka	70161
Acrylamide solution 30%	BioRad	161-0158	For gel electrophoresis
Ammonium persolphate	BioRad	161-0700	For gel electrophoresis
Glycine	BioRad	161-0718	For gel electrophoresis
SDS	BioRad	161-0302	For gel electrophoresis
TEMED	BioRad	161-0800	For gel electrophoresis
Tris	BioRad	161-0719	For gel electrophoresis
Cuvettes 0.1 cm	BioRad	1652089	For electroporation
EQUIPMENT
Centrifuge 5415R	Eppendorf
Centrifuge Allegra 21R	Beckman
Chromatography apparatus GradiFrac	Pharmacia Biotech
Gene Pulser II electroporation	BioRad
Microplate Reader 550	Biorad
MiniProtean 3 cell	BioRad
Power Supply	BioRad
Sonicator 3000	Misonix