The permission by Springer and Elsevier to reprint figures is greatly acknowledged.

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 5,000 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 &#956;l aliquots. Store the aliquots at -80 &#176;C up to 6 months.</li>
	<li>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.</li>
	<li>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 &#956;l aliquots to Petri dishes containing LB agar with the appropriate antibiotic. Incubate O/N at 37 &#176;C.</li>
	<li>Purify the transformants by streaking single colonies on Petri dishes.</li>
	<li>Prepare electro-competent cells of the primary transformants and repeat steps 1.1 to 1.5 to transform with further plasmids.</li>
	<li>Prepare glycerol stocks of the co-transformants. Transfer a single colony in LB supplemented with the appropriate antibiotics, incubate at 37 &#176;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 &#8211; 80 &#176;C.</li>
</ol>
2. Co-expression of &#945; and &#949; Subunits of E. coli DNA Polymerase III
<ol>
	<li>Transfer with a sterile loop a small amount of the appropriate strain glycerol stock (e.g., TOP10/pGOOD-&#949;243 and TOP10/pGOOD-&#949;243-&#952;/pBAD&#945;1160, 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 &#176;C O/N.</li>
	<li>Transfer, using a sterile toothpick, a single colony to 1 ml of LB-antibiotics medium. Incubate 8 hr at 37 &#176;C. Dilute the pre-culture 1:500 in fresh medium and incubate at 30 &#176;C for 15 hr.</li>
	<li>Add IPTG, arabinose, or arabinose and IPTG, 1 mM each. Incubate at 30 &#176;C for 2.5 hr. Collect the cells, and store the pellets at -20 &#176;C.</li>
	<li>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.</li>
	<li>Sonicate at 15 W for 15 sec, followed by 15 sec cooling interval (4 cycles).</li>
	<li>Centrifuge the total protein extracts at 10,000 x g for 20 min, at 4 &#176;C to recover the soluble fraction.</li>
	<li>Analyze by SDS-PAGE (12.5% acrylamide) aliquots of each soluble protein extract. To this aim, transfer 20 &#956;l of each soluble protein extract to Eppendorf tubes containing 60 &#956;l of H2O and 20 &#956;l of loading buffer (250 mM Tris-HCl pH 6.8, 10 mM &#946;-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol, 0.25% (w/v) bromophenol blue) and boil for 5 min. Load 18 &#956;l of each sample and perform the electrophoresis at 140 V for about 1.5 hr.</li>
</ol>
3. Co-expression of &#945; Subunit of Deinococcus radiodurans DNA Polymerase III and &#949; Subunit of E. coli DNA Polymerase III
<ol>
	<li>Prepare a pre-culture of the E. coli strain TOP10/pBAD-&#945;Dr/pGOOD-&#949;243 in 1 ml of LB-ampicillin-tetracycline medium and incubate 8 hr at 37 &#176;C. Dilute 1:1,000 in fresh medium and incubate O/N at 37 &#176;C.</li>
	<li>Dilute 1:100 into 200 ml of fresh medium, incubate at 37 &#176;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 &#176;C.</li>
	<li>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.</li>
	<li>Immediately centrifuge the cells lysate at 10,000 x g for 20 min. Discard the pellet. Filter the supernatant with a B&#252;chner funnel equipped with 3 layers of paper filter. Apply gentle vacuum. To avoid excessive foaming, keep the vacuum Erlenmeyer flask on ice during filtration.</li>
	<li>Determine protein concentration according to Bradford14.</li>
</ol>
4. Gel Filtration Chromatography
<ol>
	<li>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 &#176;C throughout.</li>
	<li>Collect 0.9 ml fractions and analyze them by SDS-PAGE. Transfer 20 &#956;l of each relevant fraction to Eppendorf tubes containing 60 &#956;l of H2O and 20 &#956;l of loading buffer (250 mM Tris-HCl pH 6.8, 10 mM &#946;-mercaptoethanol, 10% (w/v) SDS, 50% (v/v) glycerol, 0.25% (w/v) bromophenol blue) and boil for 5 min. Load 18 &#956;l of each sample and perform the electrophoresis at 140 V for about 1.5 hr.</li>
	<li>Determine the 3&#8217;-5&#8217; exonuclease and the DNA polymerase activity of each fraction in 96-well microplates. Assay the exonuclease activity using thymidine 5&#8217;-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.</li>
</ol>
5. Mutation Analysis of Populations Co-expressing the Wild-type &#949; Subunit of E. coli DNA Polymerase III and the Mutagenic &#949;D12A Variant
<ol>
	<li>Transfer a single colony of E. coli TOP10 containing the pBAD-&#949; and the pGOOD1-&#949;D12A11 vectors to 1 ml of LB-antibiotics medium. Incubate O/N at 37 &#176;C.</li>
	<li>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 &#176;C for 8 hr. Prepare in parallel non-induced cultures.</li>
	<li>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 &#176;C.</li>
	<li>Repeat steps 5.2 and 5.3 and collect aliquots of 1 ml.</li>
	<li>Determine the number of generations occurred in each culture. Transfer on LB plates, 100 &#956;l of appropriate serial dilutions of inoculum and culture. Incubate O/N at 37 &#176;C. Count the colonies on the LB plates, and calculate the log of the number of cells present in the inoculum (logI) and in the culture at the end of growth (logC). To determine the number of generations, use the formula: (logC&#8211; logI)/0.301.</li>
	<li>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.</li>
	<li>Determine the &#946;-glucosidase activity of each aliquot in a 96-well microplate, using p-nitrophenyl-&#946;-D-glucopyranoside (PNPGluc) as substrate. Add to each well 100 &#181;l of permeabilized cells and 100&#160;&#181;l 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.</li>
</ol>

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The authors declare that they have no competing financial interests.

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><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A9518</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>288306</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>INT</td><td>Sigma-Aldrich</td><td>I8377</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I5502</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>L-Arabinose</td><td>Sigma-Aldrich</td><td>A3256</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>31434</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>PNP-gluc</td><td>Sigma-Aldrich</td><td>N7006</td><td></td></tr><tr><td>pNP-TMP</td><td>Sigma-Aldrich</td><td>T0251</td><td></td></tr><tr><td>Tetracyclin</td><td>Sigma-Aldrich</td><td>87128</td><td></td></tr><tr><td>Trizma base&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>95039</td><td></td></tr><tr><td>Yeast extract</td><td>Fluka</td><td>70161</td><td></td></tr><tr><td>Acrylamide solution 30%</td><td>BioRad</td><td>161-0158</td><td>For gel electrophoresis</td></tr><tr><td>Ammonium persolphate</td><td>BioRad</td><td>161-0700</td><td>For gel electrophoresis</td></tr><tr><td>Glycine</td><td>BioRad</td><td>161-0718</td><td>For gel electrophoresis</td></tr><tr><td>SDS</td><td>BioRad</td><td>161-0302</td><td>For gel electrophoresis</td></tr><tr><td>TEMED</td><td>BioRad</td><td>161-0800</td><td>For gel electrophoresis</td></tr><tr><td>Tris</td><td>BioRad</td><td>161-0719</td><td>For gel electrophoresis</td></tr><tr><td>Cuvettes 0.1 cm</td><td>BioRad</td><td>1652089</td><td>For electroporation</td></tr><tr><td>Centrifuge 5415R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Centrifuge Allegra 21R</td><td>Beckman</td><td></td><td></td></tr><tr><td>Chromatography apparatus GradiFrac</td><td>Pharmacia Biotech</td><td></td><td></td></tr><tr><td>Gene Pulser II electroporation</td><td>BioRad</td><td></td><td></td></tr><tr><td>Microplate Reader 550</td><td>Biorad</td><td></td><td></td></tr><tr><td>MiniProtean 3 cell</td><td>BioRad</td><td></td><td></td></tr><tr><td>Power Supply</td><td>BioRad</td><td></td><td></td></tr><tr><td>Sonicator 3000</td><td>Misonix</td><td></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...

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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 ...

the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

Research

JoVE Journal

Biology

11.9K Views.  University of Bologna. 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. 

Video: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Genetics

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Bioengineering

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Biochemistry

Assessment of Hsp70 Chaperone Activity Using Escherichia coli dnaK756 Cells

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Author Spotlight: Exploring Heat Shock Proteins in Malaria and Tuberculosis Infections

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Immunology and Infection

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 physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

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

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

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

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

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

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

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

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

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