Name: the multifaceted benefits of protein co-expression in escherichia coli
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1. Isolation of E. coli Co-transformants
<ol>
	<li>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 &#176;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 &#176;C.</li>
	<li>At log-phase (0.6 O.D.) centrifuge the cells suspension at 5,...

<ol>
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E., Otting, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proofreading+exonuclease+subunit+&#949;+of+Escherichia+coli+DNA+polymerase+III+is+tethered+to+the+polymerase+subunit+&#945;+via+a+flexible+linker.">The proofreading exonuclease subunit &#949; of Escherichia coli DNA polymerase III is tethered to the polymerase subunit &#945; via a flexible linker.</a> Nucleic Acids Res. 36 (15), 5074-5082 (2008).</li><li>Bressanin, D., Stefan, A., Dal Piaz, F., Cianchetta, S., Reggiani, L., Hochkoeppler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+of+the+proofreading+subunit+controls+the+assembly+of+Escherichia+coli+DNA+polymerase+III+catalytic+core.">Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core.</a> Biochim. Biophys. 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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 &#949; subunit of E. coli DNA polymerase III features two distinct domains26,27, namely: i) the N-ter domain, bearing the 3&#8217;-5&#8217; exonuclease activity, and competent in binding the &#952; su...

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.,&#160;insulin). Since the advent of these technologies, significant advances have been achieved to increase the yield and quality of recombinant proteins. In addition, prokaryotic1,2 and eukaryotic3 overexpression systems were developed over the years, offering useful alternatives to the &#8220;work horse&#8221; 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. coli1,2; 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 level5,6. 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/oxygenase7,8, 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 lacIq 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., &#945; (the 5&#8217;-3&#8217; polymerase), &#949; (the 3&#8217;-5&#8217; exonuclease) and &#952; (stabilizing &#949;)13. In particular, we demonstrated that the co-expression of the &#945;&#949;&#952; 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.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agar</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1296</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">288306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">EDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">INT</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">I8377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">I5502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Arabinose</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl2</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNP-gluc</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">N7006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pNP-TMP</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T0251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetracyclin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma base&#160;</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptone</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">95039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast extract</td>
			<td style="width:125px;">Fluka</td>
			<td style="width:119px;">70161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acrylamide solution 30%</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0158</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium persolphate</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0700</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0718</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0302</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0800</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0719</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cuvettes 0.1 cm</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">1652089</td>
			<td>For electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EQUIPMENT</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge 5415R</td>
			<td style="width:125px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Allegra 21R</td>
			<td style="width:125px;">Beckman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chromatography apparatus GradiFrac</td>
			<td style="width:125px;">Pharmacia Biotech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Pulser II electroporation</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Reader 550</td>
			<td style="width:125px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MiniProtean 3 cell</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Power Supply</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator 3000</td>
			<td style="width:125px;">Misonix</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the multifaceted benefits of protein co-expression in escherichia coli

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 &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, 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 &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; 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 &#949; 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-&#946;-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.

The &#949; subunit of E. coli DNA polymerase III consists of 243 amino acids and features poor solubility17,18, unless the residues 187-243 are deleted17. However, we have previously shown11 that the co-expression of full-length &#945;, &#949;, and &#952; 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 &#945; and &#949; subunits can be in...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

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.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Research

JoVE Journal

Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments