This work was funded by the Israel Science Foundation's Center for Research Excellence (I-CORE) in Structural Cell Biology (1775/12) and the Israel Science Foundation grants 1721/16 and 2338/16 (Y.H.), and 825/14 (D.K.). The support of the Fields Estate Foundation to D.K. is highly appreciated. This work was performed by Ilan Edri and Michal Goldenberg in partial fulfillment of the M.D. thesis requirements of the Sackler Faculty of Medicine, Tel Aviv University.

1 . Cloning of cis-PT for Overexpression in E. coli
<ol>
	<li>Obtain pET-32b expression vector, designed for cloning and high-level expression of protein sequences fused with the 109aa thioredoxin (TRX) protein 17, and E. coli codon-optimized 18 coding sequence of full-length cis-PT.</li>
	<li>Take care to have a TEV-protease (tobacco etch virus protease) cleavage site (ENLYFQ/G, where &#34;/&#34; indicates the cleavage point) 9 followed by a short flexible linker (SGSGSG, to enhance cleavage site accessibility) upstream of the cis-PT sequence (Figure 1A). Also, add suitable 5&#39; and 3&#39; restriction sites for cloning.</li>
	<li>Clone the synthesized DNA construct into the pET-32b vector using standard ligation techniques 10.</li>
</ol>
2 . Overexpression of Human DHDDS in E. coli
<ol>
	<li>Transform E. coli T7 express competent cells with the TRX-fusion DHDDS construct according to the manufacturer&#39;s instructions.</li>
	<li>Plate the transformed cells according to the manufacturer&#39;s instructions on LB-agar under ampicillin selective conditions (200 mg/L).</li>
	<li>Inoculate selected colonies into 100 mL of LB medium in 250 mL flasks starter cultures with 100 mg/L ampicillin and incubate overnight at 37 &#176;C with constant shaking at 200 rpm.</li>
	<li>Inoculate 80 mL of the culture into two 5 L flasks (40 mL per flask), each containing 1.5 L of 2x YT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) with 100 mg/L ampicillin.</li>
	<li>Incubate the culture at 37 &#176;C with constant shaking at 180 rpm until reaching OD600nm = 0.5.</li>
	<li>Lower the temperature to 16 &#176;C and induce the cells by adding 0.5 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG). Continue incubating under the same condition for 16-20 h for protein expression.</li>
	<li>Harvest the cells by centrifugation (10,000 x g for 10 min). 
	Note: The protocol can be paused here; the cells pellet should be kept at -80&#176;C until purification.</li>
</ol>
3 . Purification of Human DHDDS
<ol>
	<li>Resuspend the cells in buffer A (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10 mM &#946;-mercaptoethanol), 1 &#181;g/mL DNase I, and a protease inhibitor mixture.</li>
	<li>Homogenize the cells using a glass-Teflon homogenizer.</li>
	<li>Disrupt the cells using a microfluidizer or equivalent at 12,000 - 15,000 psi 11.</li>
	<li>Centrifuge the cell lysate at 40,000 x g for 45 min at 4 &#176;C. Recover the supernatant, containing the soluble fraction.</li>
	<li>Load the supernatant onto a 5 - 10 mL cobalt immobilized-metal affinity chromatography (Co2+-IMAC)12 column with 10 mM imidazole, followed by thorough washing with buffer A and 10 mM imidazole to reduce nonspecific protein binding.</li>
	<li>Elute the overexpressed proteins with buffer A supplemented with 250 mM imidazole.</li>
	<li>Remove imidazole using a 53 mL preparative desalting column equilibrated with buffer A.</li>
	<li>Add 6xHis-tagged TEV protease (1 mg TEV protease per 50 mg protein) to the eluted proteins to remove their 6xHis-tagged TRX fusion protein at 4 &#176;C overnight.</li>
	<li>Load the cleaved proteins onto a Co2+-IMAC column, equilibrated with buffer A supplemented with 5 mM imidazole, to remove the cleaved 6x His-tagged TRX fusion protein and TEV protease.</li>
	<li>Collect the flow-through and concentrate to 3 - 4 mL using a 30 kDa molecular weight cut-off centrifugal filter.</li>
	<li>Load the concentrated protein onto a superdex-200 size-exclusion chromatography column equilibrated with buffer A for final purification. The protein elutes as a dimer (~76 kDa).</li>
	<li>Select relevant fractions by comparing the elution profile to a column calibration curve.</li>
	<li>Assess the purity of the preparation using SDS-PAGE.</li>
	<li>Determine protein concentration needed for downstream experimentation and flash-freeze aliquots of purified, concentrated proteins in liquid nitrogen and store at -80 &#176;C until use.</li>
</ol>
4 . Analytical Size-exclusion Chromatography (SEC)
<ol>
	<li>To ensure the ability of the protein to endure freeze-thaw cycles, thaw an aliquot of the frozen proteins and centrifuge at 21,000 x g for 10 min to remove insoluble aggregates. Collect the supernatant.</li>
	<li>Load the proteins onto a superdex-200 analytical column pre-equilibrated with 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.02% Triton X-100, 10 mM &#946;-mercaptoethanol, using an ultra-performance liquid chromatography system 13,14.</li>
	<li>Monitor tryptophan fluorescence (&#955;ex = 280 nm, &#955;em = 350 nm) to detect the proteins&#39; eluting elution profile. Be sure to have a valid calibration curve for molecular weight assessment - such curves are available via the SEC column manufacturers.</li>
</ol>
5 . Enzyme Kinetics - Time-dependent Activity 15,16,17
<ol>
	<li>To ensure the activity of the purified protein, mix 5 &#956;M of purified DHDDS with 10 &#956;M FPP and 50 &#956;M 14C-IPP to initiate the reaction in buffer A with 0.5 mM MgCl2 at 22 - 30 &#176;C. 
	Note: The exact reaction conditions may vary with preparations of cis-PTs different from DHDDS. Moreover, the activity of DHDDS also depends on the temperature and [Mg2+]. 
	Caution: 14C-IPP is radioactive and should be used according to local radiation safety regulations.</li>
	<li>Withdraw 15 &#956;L samples from the reaction at 0, 2, 4 and 6 h, following immediate quenching of the reaction by the addition of 15 &#956;L buffer A supplemented 20 mM EDTA (to a final concentration of 10 mM EDTA).</li>
	<li>Add 1 mL H2O-saturated 1-butanol and vortex thoroughly to extract the reaction products. 
	Note: The protocol can be stopped here and the samples can be kept for later reading.</li>
	<li>Add scintillation cocktail to the samples and, using a scintillation counter, quantitate reaction products in the butanol phase encompassing 14C, together with the radioactivity of 15 &#956;L from the reaction mixture, representing the total radioactivity.</li>
	<li>Subtract the background readings measured using the 0 h samples from each time point and calculate the percent of 14C-IPP utilized.</li>
	<li>Calculate the net 14C-IPP incorporation at each time point by calculating the percent utilized from the total 14C-IPP concentrations, and plot the results as a function of time. An increase in net 14C-IPP incorporation with time is expected.</li>
</ol>

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E., Cherry, S., Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+soluble+His(6)-tagged+TEV+protease.">Expression and purification of soluble His(6)-tagged TEV protease.</a> Methods Mol Biol. 498, 297-307 (2009).</li><li>Crouse, G. F., Frischauf, A., Lehrach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+and+simplified+approach+to+cloning+into+plasmids+and+single-stranded+phages.">An integrated and simplified approach to cloning into plasmids and single-stranded phages.</a> Methods Enzymol. 101, 78-89 (1983).</li><li>Middelberg, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process-scale+disruption+of+microorganisms.">Process-scale disruption of microorganisms.</a> Biotechnol Adv. 13 (3), 491-551 (1995).</li><li>Block, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilized-metal+affinity+chromatography+(IMAC):+a+review.">Immobilized-metal affinity chromatography (IMAC): a review.</a> Methods Enzymol. 463, 439-473 (2009).</li><li>Sachyani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+a+Kv7.1+potassium+channel+gating+module:+studies+of+the+intracellular+c-terminal+domain+in+complex+with+calmodulin.">Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin.</a> Structure. 22 (11), 1582-1594 (2014).</li><li>Shuart, N. G., Haitin, Y., Camp, S. S., Black, K. D., Zagotta, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+for+3:1+subunit+stoichiometry+of+rod+cyclic+nucleotide-gated+ion+channels.">Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels.</a> Nat Commun. 2, 457 (2011).</li><li>Chang, S. Y., Tsai, P. C., Tseng, C. S., Liang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refolding+and+characterization+of+a+yeast+dehydrodolichyl+diphosphate+synthase+overexpressed+in+Escherichia+coli.">Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.</a> Protein Expr Purif. 23 (3), 432-439 (2001).</li><li>Chang, S. Y., Ko, T. P., Liang, P. H., Wang, A. 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The authors have nothing to disclose.

The protocol described here for purification of functional human DHDDS in E. coli cells is simple and efficient, allowing one to overexpress and purify the protein in 3 - 4 days once a suitable construct is available. Such protocols for protein purification are of special significance given the breakthroughs in genome sequencing, which provided plethora of information regarding the genetics of many diseases 18, thereby requiring the development of high-throughput methods to characterize p...

Prenyltransferases are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions 1,2. Z-type enzymes catalyze the formation of cis double bonds from the condensation reaction, whereas E-type enzymes catalyze trans double bond formation 3. cis-Prenyltransferases (cis-PT, Z-type enzymes) are classically classified according to their product chain length into short-chain (C15), medium-chain (C50-55), and long-chain (C70-120) 4. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP) 1,5,6. This results in the formation of dehydrodolichyl diphosphate, a C55-100polyprenyl diphosphate serving as a precursor for dolichylpyrophosphate, the glycosyl carrier molecule involved in N-linked protein glycosylation 1. Among Ashkenazi Jews, a missense non-conservative mutation (K42E) in DHDDS results in autosomal recessive retinitis pigmentosa 7,8. Therefore, the present protocol was developed in order to acquire purified DHDDS suitable for mechanistic studies.
Escherichia coli is considered the most convenient and cost-effective host for recombinant protein expression, and is therefore also the most frequently used host. However, when one attempts to heterologously overexpress proteins in E. coli, protein-specific considerations should be made. Obtaining properly folded, active recombinant proteins from E. coli, is not a simple matter due to the distinct properties of different proteins. Numerous approaches have been developed to overcome these hurdles. 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. Of note, a previous attempt to overexpress yeast cis-PT without protein fusion was unsuccessful due to complete insolubility even in the presence of detergent 12. The described protocol is simple, cost-effective, time sparing and allows one to obtain DHDDS preparations suitable for mechanistic studies. Given the homology of cis-PT among different species, we suggest that this protocol may be applied for other eukaryotic cis-PT as well.

<table><tbody><tr><td>pET-32b</td><td>Novagen</td><td>69016-3</td><td></td></tr><tr><td>T7 Express&amp;nbsp;lysY&amp;nbsp;Competent&amp;nbsp;&lt;em&gt;E. coli&lt;/em&gt;&amp;nbsp;(High Efficiency)</td><td>NEB</td><td>C3010I</td><td></td></tr><tr><td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>TALON-superflow resin</td><td>GE Healthcare</td><td>28-9574-99</td><td></td></tr><tr><td>HiPrep 26/10 desalting column&amp;nbsp;</td><td>GE Healthcare</td><td>17508701</td><td></td></tr><tr><td>HiLoad 16/60 superdex-200&amp;nbsp;</td><td>GE Healthcare</td><td>28989335</td><td></td></tr><tr><td>superdex-200 increase 5/150 GL&amp;nbsp;</td><td>GE Healthcare</td><td>28990945</td><td></td></tr><tr><td>&lt;sup&gt;14&lt;/sup&gt;C-Isopentenyl pyrophosphate</td><td>Perkin-Elmer</td><td>NEC773050UC&amp;nbsp;</td><td></td></tr><tr><td>trans,trans-Farnesyl pyrophosphate</td><td>Sigma</td><td>44270-10MG</td><td></td></tr></tbody></table>

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.

General overview of the construct used here and the purification process are shown in Figure 1. The samples obtained at each purification step are shown in Figure 2. This SDS-PAGE analysis shows the stepwise purification of DHDDS, resulting in a highly purified product. Figure 3 shows the results of analytical SEC of the purified enzyme, revealing that the protein is only observed as a homodimer. <st...

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

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.

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 ...

overexpression-purification-human-cis-prenyltransferase-escherichia

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JoVE Journal

Immunology and Infection

6.6K Views.  Tel Aviv University.  Een eenvoudig protocol voor overexpressie en zuivering van codon-geoptimaliseerde, humane cis -prenyltransferase, onder niet-denaturerende omstandigheden, van Escherichia Coli , wordt beschreven, samen met een enzymatische activiteitstest. Dit protocol kan worden genegaliseerd voor de productie van andere cis- prenyltransferase eiwitten in hoeveelheid en kwaliteit geschikt voor mechanistische studies. 

Video: Overexpression and Purification of Human Cis-prenyltransferase 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

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biochemistry

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

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

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Biology

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 ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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