The authors thank the NIH (AI074224 and GM085323) and NSF (0712019 and 0924699) for funding to support projects dedicated to heterologous biosynthesis.

The text below is specific for the erythromycin A antibiotic, but the steps are designed to be generally applicable to other natural products as candidates for heterologous biosynthesis.
1. Erythromycin A Genetic Cluster Transfer
<ol>
	<li>Design PCR primers to amplify all of the genes associated with the erythromycin A cluster within the S. erythraea chromosome. This step is specific to those natural product candidates whose genetic sequences have been determined. Alternatively, the required genes may be synthesized (through several commercial vendor options) with the advantage that the newly synthesized genes would be designed for optimal codon usage within the new E. coli host. If DNA synthesis is pursued, a current challenge is generating megasynthase genes which may exceed 10 kb in length. Technically, synthesis can be accomplished, but it is challenging and costly. It is anticipated that continual improvements in gene synthesis technology will address current drawbacks and further validate this approach for providing heterologous genetic material.</li>
	<li>Perform PCR using freshly prepared S. erythraea genomic DNA as a template. The chromosomal DNA may be prepared from cultures of S. erythraea or from frozen stocks of the bacterium (Figure 3)25. PCR reactions may benefit from the addition of DMSO (4 &mu;l in 50 &mu;l reactions) to account for high G+C content in the target S. erythraea genes. Each amplified gene should be sequenced to confirm that the full gene has been retrieved and that no mutations have been introduced during the PCR process.</li>
	<li>Through restriction digest and ligation, insert each of the isolated genes into expression vectors designed for E. coli (Figure 3). This is initiated by designing restriction sites within the PCR primers to allow digestion and ligation into suitable expression vectors (an example has been included in Figure 3). We have used both pET21 and pET28 vectors. The pET28 vector allows the inclusion of a 5' leader sequence which aided gene expression in the erythromycin A case. Confirm individual gene expression through RT-PCR, SDS-PAGE analysis (using soluble protein fractions), or convenient phenotypic assays (Figure 4).</li>
	<li>Construct operons based upon genes successfully expressed from individual expression plasmids (Figure 3). We have traditionally used combinations of compatible-cohesive restriction enzymes (such as XbaI and SpeI) 24 to sequentially convert genes to operons; however, the restriction sites chosen for operon construction must not reside within (or must be removed from) the isolated gene sequences (an issue that can be avoided if gene synthesis is used). This step consolidates the 20 erythromycin A genes prior to E. coli transformation. Currently, we have used this method to include up to 20 kb of foreign DNA into a standard pET vector (two genes of ~10 kb each). Furthermore, we have used this approach to generate operons containing nine genes. It is unknown exactly how much DNA or how many genes may be integrated into pET-type plasmids; however, the metrics cited above provide a reference for complicated systems like the erythromycin biosynthetic pathway. Finally, standard in vitro ligation steps and subsequent successful transformation steps become increasingly difficult as operon and plasmid size increases. Ligation temperature (12-22 &deg;C) and time (3-24 hr) are varied and electro-transformation is used to aid the process of producing final plasmid constructs.</li>
	<li>Transform the newly designed biosynthetic plasmids into E. coli strain BAP1 using electro-transformation (Figure 5). The procedure may be attempted with plasmids introduced either sequentially or in combination. When transforming size-able or numerous plasmids, we have traditionally used electro-transformation (as opposed to chemical transformation). Once transformed, plate the cells on solid LB medium containing tetracycline (5 mg/l), carbenicillin (100 mg/l), kanamycin (50 mg/l), streptomycin (50 mg/l), and apramycin (50 mg/l) to select for plasmid maintenance. It is highly unusual to use six different selection antibiotics post-transformation of an E. coli cell, but this simply reflects the total number of plasmids needed to allow final erythromycin A biosynthesis. While possible, the approach will limit the production capabilities of the compound once culturing has begun (please see below).</li>
</ol>
2. E. coli Biosynthetic Reconstitution
<ol>
	<li>Strain BAP1 has been designed to provide the required substrates for biosynthesis and to post-translationally modify the deoxyerythronolide B megasynthase. Individual genes of the erythromycin A pathway have been tested for gene expression and consolidated as operons into expression plasmids. This section is dedicated to culture conditions to test for concerted activity of the full erythromycin A pathway.</li>
	<li>Take 3 separate colonies of the freshly transformed BAP1 and inoculate 1.5 ml LB cultures containing the required plasmid selection antibiotics (at the same concentrations indicated above to select for transformants on solid medium), Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG; 100 &mu;M) and arabinose (2 mg/ml) to induce gene expression, and 20 mM sodium propionate to support intracellular biosynthetic precursor formation. Once production has been confirmed, larger cultures may be conducted in an attempt to scale and maximize product titers.</li>
	<li>Culture the cells at 22 &deg;C and 250 rpm for 24-48 hr. In this case, antibiotic selection ensures that the required plasmids are maintained in the early stages of cellular culture. However, plasmids may be easily lost as selection antibiotic levels change over the course of the culturing period. This may lead to plasmid instability (i.e., the loss of one or more plasmids during culture), which would then limit production capability. The problem is exacerbated when incompatible plasmids are used to introduce a complete heterologous gene cluster. Such is the case for the erythromycin A compound (Figure 5) and would have to be remedied to truly overproduce the compound from E. coli. However, for the purpose of demonstrating initial production, such design flaws are tolerated for the sake of expeditiously establishing the feasibility of the heterologous approach.</li>
	<li>Clarify the cultures by centrifugation at 10,000 rpm in an Eppendorf microfuge. Remove the supernatant and store at 4 &deg;C for analysis.</li>
	<li>In the case of erythromycin A, lack of compound production was addressed by the inclusion of the GroES/EL chaperonin (using pGro7 specifically to address lack of activity of the enzymes post-expression) and the inclusion of additional gene copies (for those genes suspected of weak expression/activity). Similar strategies may be needed with other heterologous natural product production efforts.</li>
</ol>
3. Product Analysis
<ol>
	<li>To confirm and quantify erythromycin A formation, inject the culture supernatant onto an LC-MS system (Figure 6). Commercially available erythromycin A was used to confirm production and as a standard during quantification. For cases without commercially available standards, LC-MS analysis would require additional chemical characterization by NMR and high resolution mass spectrometry.</li>
	<li>Compare production between the 3 colonies/cultures from the BAP1 transformation. Choose the highest producer and prepare a glycerol stock from the colony source. This step can be done in parallel with the production cultures of Protocol Section 2 by using a small portion of the cultures to prepare glycerol stocks. Store the glycerol stock in a -80 &deg;C freezer.</li>
	<li>Using the confirmed, highest producing cell stock, begin a separate production culture and extract the final supernatant with 2x volume ethyl acetate. Use a vacuum evaporative system to dry the extract and resuspend in 100 &mu;l methanol. Transfer the extract to a sterile filter disk (~1/4 inch diameter) and allow the disk to dry. Separately, culture B. subtillis in LB liquid overnight. Add 20 &mu;l of the B. subtilis culture to 20 ml of liquid LB agar at 45 &deg;C. Plate the agar and allow to solidify. Place the filter disk on the plate and incubate at 37 &deg;C (Figure 7). Alternatively, the extract can be added to liquid cultures within a 96-well plate to quantify the activity using a plate reader (Figure 7).</li>
</ol>

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No conflicts of interest declared.

Critical steps in heterologous biosynthesis are encountered at each of the three procedural points in the process: 1) genetic transfer; 2) biosynthetic reconstitution; and 3) product analysis. A problem at any stage will derail the ultimate objective of establishing heterologous biosynthesis. Perhaps the most challenging aspect of the process is establishing reconstituted biosynthesis, since this is absolutely required to allow successful analysis. However, reconstitution is dependent upon careful design and transfer of ...

Erythromycin A is a polyketide antibiotic produced by the Gram-positive soil bacterium Saccharopolyspora erythraea, and current production has been incrementally improved to ~10 g/L through decades of traditional mutagenesis and screening protocols and more recently through process optimization schemes 1-6. Mutagenesis and screening strategies are common in antibiotic natural product development as a result of difficulties in culturing and/or genetically manipulating native production hosts and because of the readily available antibiotic activity or improved growth phenotypes to aid selection. In the case of erythromycin A, S. erythraea is limited by a slow growth profile and the lack of more direct genetic manipulation techniques (relative to organisms like E. coli), thus, hampering rapid improvements in production and the biosynthesis of new derivatives. Having recognized the production issues and unlocked diversification possibilities with compounds like erythromycin A, the research community began to pursue the idea of heterologous biosynthesis (Figure 1) 7. These efforts coincided with available sequence information for the erythromycin A gene cluster 8-11. It should be emphasized that the number of sequenced complex natural product gene clusters has greatly expanded 12-16, providing the impetus for continued efforts in heterologous biosynthesis to access encoded medicinal potential. To do so, heterologous reconstitution requires that the new host meet the needs of the specific biosynthetic pathway. E. coli provides technical convenience, a wide-spanning set of molecular biology techniques, and metabolic and process engineering strategies for product development. Yet, when compared to native production hosts, E. coli does not exhibit the same level of complex natural product production. It was therefore unknown whether E. coli could serve as a viable heterologous option for complex natural product biosynthesis. However, it was assumed that E. coli would be an ideal host organism if heterologous biosynthesis could be accomplished. 
With this goal in mind, initial efforts began to produce the polyketide aglycone 6-deoxyerythronolide B (6dEB) through E. coli. However, native E. coli metabolism could not provide appreciable levels of the propionyl-CoA and (2S)-methylmalonyl-CoA precursors needed to support 6dEB biosynthesis nor could the new host post-translationally modify the deoxyerythronolide B synthase (DEBS) enzymes. To remedy these issues, a metabolic pathway composed of native and heterologous enzymes was built into E. coli such that exogenously fed propionate was converted intracellularly to propionyl-CoA and then (2S)-methylmalonyl-CoA; during the engineering to complete this pathway, an sfp gene was placed into the chromosome of E. coli BL21(DE3) to produce a new strain termed BAP1. The Sfp enzyme is a phosphopantetheinyl transferase capable of attaching the 4'-phosphopantetheine cofactor to the DEBS enzymes 17,18. The three DEBS genes (each ~10 kb) were then placed on two separately selectable expression vectors containing inducible T7 promoters. After a key adjustment of post-induction temperature (to 22 &deg;C), the DEBS genes were coordinately expressed within BAP1 in an active state capable of generating 6dEB 19. 
 The pursuit of full erythromycin A biosynthesis then began using an analogous gene cluster from Micromonospora megalomicea or a hybrid pathway composed of genes from S. erythraea, S. fradiae, and S. venezuelae which produced the intermediates erythromycin C and 6-deoxyerythromycin D, respectively 20-22. Recently, our group has extended these efforts by producing erythromycin A (the most clinically-relevant form of erythromycin) through E. coli. In contrast to previous work, our strategy coordinately expressed the 20 original S. erythraea genes needed for polyketide biosynthesis, deoxysugar biosynthesis and attachment, additional tailoring, and self-resistance (Figure 2). In total, 26 (native and heterologous) genes were engineered to allow E. coli to produce erythromycin A at 4 mg/L 23,24. This result established complete production of a complex polyketide natural product using E. coli and serves as a basis to leverage this new production option or pursue new ones.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>PCR machine</td>
 <td>Eppendorf</td>
 <td>Mastercycler personal</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>Fisher</td>
 <td>BP231</td>
 <td></td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>BioRad</td>
 <td>Micropulser</td>
 <td></td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Fisher</td>
 <td>BP1620</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium propionate</td>
 <td>Sigma</td>
 <td>P1880</td>
 <td></td>
 </tr>
 <tr>
 <td>L-arabinose</td>
 <td>Sigma</td>
 <td>A3256</td>
 <td></td>
 </tr>
 <tr>
 <td>Refrigerated Shaker</td>
 <td>Thermo Scientific</td>
 <td>MaxQ 4000</td>
 <td></td>
 </tr>
 <tr>
 <td>Microfuge</td>
 <td>Eppendorf</td>
 <td>Centrifuge 5415D</td>
 <td></td>
 </tr>
 <tr>
 <td>pGro7</td>
 <td>Takara</td>
 <td>Chaperone Plasmid Set (3340)</td>
 <td></td>
 </tr>
 <tr>
 <td>pET21, pET28, pCDF-Duet-1</td>
 <td>EMD Chemicals</td>
 <td>69742-3, 69864, 71340</td>
 <td></td>
 </tr>
 <tr>
 <td>LC-MS</td>
 <td>Applied Biosystems</td>
 <td>3200 Q-Trap</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl acetate</td>
 <td>Sigma</td>
 <td>270989</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Sigma</td>
 <td>322415</td>
 <td></td>
 </tr>
 <tr>
 <td>Vacuum centrifuge</td>
 <td>Eppendorf</td>
 <td>Concentrator 5301</td>
 <td></td>
 </tr>
 <tr>
 <td>Rotary Evaporator</td>
 <td>Buchi</td>
 <td>R-200</td>
 <td></td>
 </tr>
 </tbody>
</table>

the logic, experimental steps, and potential of heterologous natural product biosynthesis featuring the complex antibiotic erythromycin a produced through e. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The desired outcome of this approach is the production of a fully bioactive natural product from the E. coli heterologous host. This is best represented by the LC-MS results used to confirm and quantify production (Figure 6) and the antibacterial bioassay used to confirm final activity (Figure 7). In the overall scheme of heterologous biosynthesis, this result defines success. Once accomplished, research efforts then turn to optimization (both at the cellular and process s...

Watch this Scientific Journal Video about The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. 

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

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Research

JoVE Journal

Biology

18.4K Views.  State University of New York at Buffalo. The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

Video: The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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 the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

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

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

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

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...