Wheat Germ System Research Articles

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology.

Cell-free protein synthesis (CFPS) systems are gaining more importance as universal tools for basic research, applied sciences, and product development with new technologies emerging for their application. Huge progress was made in the field of synthetic biology using CFPS to develop new proteins for technical applications and therapy. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields with the use of a eukaryotic ribosome, making it an excellent approach for the synthesis of complex eukaryotic proteins including, for example, protein complexes and membrane proteins. Separating the translation reaction from other cellular processes, CFPS offers a flexible means to adapt translation reactions to protein needs. There is a large demand for such potent, easy-to-use, rapid protein expression systems, which are optimally serving protein requirements to drive biochemical and structural biology research. We summarize here a general workflow for a wheat germ system providing examples from the literature, as well as applications used for our own studies in structural biology. With this review, we want to highlight the tremendous potential of the rapidly evolving and highly versatile CFPS systems, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

Protein structural biology using cell-free platform from wheat germ

One of the biggest bottlenecks for structural analysis of proteins remains the creation of high-yield and high-purity samples of the target protein. Cell-free protein synthesis technologies are powerful and customizable platforms for obtaining functional proteins of interest in short timeframes, while avoiding potential toxicity issues and permitting high-throughput screening. These methods have benefited many areas of genomic and proteomics research, therapeutics, vaccine development and protein chip constructions. In this work, we demonstrate a versatile and multiscale eukaryotic wheat germ cell-free protein expression pipeline to generate functional proteins of different sizes from multiple host organism and DNA source origins. We also report on a robust purification procedure, which can produce highly pure (> 98%) proteins with no specialized equipment required and minimal time invested. This pipeline successfully produced and analyzed proteins in all three major geometry formats used for structural biology including single particle analysis with electron microscopy, and both two-dimensional and three-dimensional protein crystallography. The flexibility of the wheat germ system in combination with the multiscale pipeline described here provides a new workflow for rapid production and purification of samples that may not be amenable to other recombinant approaches for structural characterization.

Cucumber mosaic virus coat protein modulates the accumulation of 2b protein and antiviral silencing that causes symptom recovery in planta.

Shoot apical meristems (SAM) are resistant to most plant viruses due to RNA silencing, which is restrained by viral suppressors of RNA silencing (VSRs) to facilitate transient viral invasion of the SAM. In many cases chronic symptoms and long-term virus recovery occur, but the underlying mechanisms are poorly understood. Here, we found that wild-type Cucumber mosaic virus (CMVWT) invaded the SAM transiently, but was subsequently eliminated from the meristems. Unexpectedly, a CMV mutant, designated CMVRA that harbors an alanine substitution in the N-terminal arginine-rich region of the coat protein (CP) persistently invaded the SAM and resulted in visible reductions in apical dominance. Notably, the CMVWT virus elicited more potent antiviral silencing than CMVRA in newly emerging leaves of infected plants. However, both viruses caused severe symptoms with minimal antiviral silencing effects in the Arabidopsis mutants lacking host RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) or SUPPRESSOR OF GENE SILENCING 3 (SGS3), indicating that CMVWT induced host RDR6/SGS3-dependent antiviral silencing. We also showed that reduced accumulation of the 2b protein is elicited in the CMVWT infection and consequently rescues potent antiviral RNA silencing. Indeed, co-infiltration assays showed that the suppression of posttranscriptional gene silencing mediated by 2b is more severely compromised by co-expression of CPWT than by CPRA. We further demonstrated that CPWT had high RNA binding activity leading to translation inhibition in wheat germ systems, and CPWT was associated with SGS3 into punctate granules in vivo. Thus, we propose that the RNAs bound and protected by CPWT possibly serve as templates of RDR6/SGS3 complexes for siRNA amplification. Together, these findings suggest that the CMV CP acts as a central hub that modulates antiviral silencing and VSRs activity, and mediates viral self-attenuation and long-term symptom recovery.

Performance benchmarking of four cell‐free protein expression systems

Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

BackgroundThe Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts.ResultsTo overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system.ConclusionWe present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

Wheat germ systems for cell-free protein expression.

Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

Expression of Functional Plasmodium falciparum Enzymes Using a Wheat Germ Cell-Free System

One decade after the sequencing of the Plasmodium falciparum genome, 95% of malaria proteins in the genome cannot be expressed in traditional cell-based expression systems, and the targets of the best new leads for antimalarial drug discovery are either not known or not available in functional form. For a disease that kills up to 1 million people per year, routine expression of recombinant malaria proteins in functional form is needed both for the discovery of new therapeutics and for identification of targets of new drugs. We tested the general utility of cell-free systems for expressing malaria enzymes. Thirteen test enzyme sequences were reverse amplified from total RNA, cloned into a plant-like expression vector, and subjected to cell-free expression in a wheat germ system. Protein electrophoresis and autoradiography confirmed the synthesis of products of expected molecular masses. In rare problematic cases, truncated products were avoided by using synthetic genes carrying wheat codons. Scaled-up production generated 39 to 354 μg of soluble protein per 10 mg of translation lysate. Compared to rare proteins where cell-based systems do produce functional proteins, the cell-free yields are comparable or better. All 13 test products were enzymatically active, without failure. This general path to produce functional malaria proteins should now allow the community to access new tools, such as biologically active protein arrays, and lead to the discovery of new chemical functions, structures, and inhibitors of previously inaccessible malaria gene products.

Translating experience: Thinking outside the box

Tony Hunter is Director of the Salk Institute Cancer Center, 10010 N Torrey Pines Road, San Diego, California 92037, USA. e-mail: hunter@salk.edu acetyl group. Had we been primed to think outside the box, we might have discovered this initiation mechanism sooner. In 1970, Richard and I were using rabbit reticulocytes to analyse the fate of nascent globin chains released prematurely from ribosomes following puromycin treatment. We found that these chains were rapidly degraded; this also occurred in vitro when isolated ribosomes incubated with puromycin were added to a reticulocyte lysate supplemented with ATP. Just before I left for my postdoctoral position at the Salk Institute, we presented these results at a 1971 meeting and, in a proceedings chapter, speculated that proteases unable to recognize intact folded globin chains degraded these truncated proteins. Had we continued the project and thought of alternative explanations, we might have uncovered the ATP-dependent protease activity, reported by Fred Goldberg in 1977 and later shown to be due to the proteasome, and the ubiquitin-mediated protein degradation system, reported by Avram Hershko and Aaron Ciechanover in 1978 — both using rabbit reticulocyte lysates. In 1973, when I returned to the Department of Biochemistry in Cambridge following my postdoc, a new willingness to think laterally led to an unexpected discovery. When a fire destroyed our lab in June 1974, we became refugees and were graciously offered temporary space in the New Addenbrookes site, opposite the MRC Laboratory of Molecular Biology (LMB). Our plight led Max Perutz, the Director of LMB, to offer us dining rights in the LMB cafeteria, where we ate lunch every day. The unspoken rule of the cafeteria was that you had to sit with people from other groups, and this soon led to Tim Hunt and myself collaborating with John Knowland and David Zimmern in the LMB tobacco mosaic virus (TMV) group, led by Aaron Klug. At the time, the packaged TMV genomic RNA was known to encode the 18 kDa viral coat protein, but all attempts to translate coat protein in vitro had failed, generating instead much larger 140–160 kDa products. We found the same when we translated TMV virion RNA in the wheat germ system we were using to study protein synthesis initiation, or in Xenopus laevis oocytes. Eventually, we realized that as TMV-infected tobacco plants make large amounts of coat protein, their leaves must contain a coat protein mRNA. One of the first experiments we did in our new home was to translate total RNA isolated from TMV-infected tobacco leaves using wheat germ samples stored in a liquid N2 freezer that had survived the fire. Gratifyingly, we obtained authentic coat protein, and demonstrated that its mRNA corresponded to the 750 bases at the 3' end of the genomic RNA. We had discovered that TMV expresses its coat protein through a subgenomic viral RNA, a strategy commonly adopted by RNA viruses for expressing individual proteins from polycistronic RNAs. Our findings did not explain why fragments from the 3' end of the genomic RNA failed to translate coat protein. This mystery was solved in 1975, when a 5' methylated cap structure was shown to be required for eukaryotic mRNA translation. This initiation mechanism restricts translation to the cistron adjacent to the cap, explaining why internal cistrons, such as the coat protein gene, cannot be translated. We now know that the virion RNA has a cap, as does the subgenomic RNA made in infected cells. These early experiences stood me in good stead when I fortuitously stumbled across a novel phospho-amino acid generated in an in vitro kinase assay using polyoma virus middle T antigen. This was not the expected phospho-serine or phospho-threonine, and only by being willing to ignore dogma did I surmise that this might be phospho-tyrosine. The rest, as they say, is history.

Cell‐free protein synthesis and purification of human dopamine D2 receptor long isoform

The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell-free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell-free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate-based system and the wheat-germ lysate-based system. The bacterial cell-free method used pET 100/D-TOPO vector to synthesize hexa-histidine-tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel-nitrilotriacetic acid affinity resin. The wheat germ system used pEU-glutathione-S-transferase (GST) vector to synthesize GST-tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in-gel protein sequencing via Nano LC-MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor.

In VitroProtein Import of a Putative Amino Acid Transporter fromArabidopsis thalianainto Chloroplasts and Its Suborganellar Localization

We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.

Rapid Colorimetric Testing for Pyrazinamide Susceptibility of M. tuberculosis by a PCR-Based In-Vitro Synthesized Pyrazinamidase Method

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays.

In vitro production of an active neurotrophic factor, neuregulin-1: Qualitative comparison of different cell-free translation systems

The individual biological activities of many neurotrophic factors and their variants, which are produced by alternative splicing and proteolytic processing, often remain to be characterized. Bacterial protein production combined with protein refolding and purification is a conventional procedure to obtain active neurotrophic factors; however, the procedure is time consuming and appropriate protein refolding in vitro is sometimes unpredictable. Here we examined three distinct cell-free translation systems: reticulocyte lysate, Hela cell lysate and wheat germ extract, which may allow us to produce biologically active factors in a single tube. Taking type-I neuregulin-1 beta3 as an example, we produced neuregulin-1 protein from its mRNAs flanked by Cap nucleotide and/or internal ribosome entry site (IRES) and compared the yields and biological activity of translation products from these systems. The protein yield from IRES+ mRNA was highest in the Hela cell-free system, while background translation was lowest in the wheat germ system. The biological activity of both translation products was modest or negligible, however. Neuregulin-1 protein was produced in reticulocyte lysate at yields of 19pmol/mL (∼500ng/mL); furthermore, it was potent at phosphorylating ErbB4 receptor and able to bind to heparin sulfate. These results demonstrate that the reticulocyte lysate translation system produces active neurotrophic factors in vitro and is useful for radiolabeling or preliminary assessment of novel neurotrophic factors and their variants.

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k(on)) and dissociation rate constant (k(off)) for eIF4F binding to IRES were 59 +/- 2.1 micro s(-1) and 12.9 +/- 0.3 s(-1), respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F approximately 3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.

The Membrane-binding Motif of the Chloroplast Signal Recognition Particle Receptor (cpFtsY) Regulates GTPase Activity

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.

A systematic approach for testing expression of human full-length proteins in cell-free expression systems.

BackgroundThe growing field of proteomics and systems biology is resulting in an ever increasing demand for purified recombinant proteins for structural and functional studies. Here, we show a systematic approach to successfully express a full-length protein of interest by using cell-free and cell-based expression systems.ResultsIn a pre-screen, we evaluated the expression of 960 human full-length open reading frames in Escherichia coli (in vivo and in vitro). After analysing the protein expression rate and solubility, we chose a subset of 87 plasmids yielding no protein product in E. coli in vivo. These targets were subjected to a more detailed analysis comparing a prokaryotic cell-free E. coli system with an eukaryotic wheat germ system. In addition, we determined the expression rate, yield and solubility of those proteins. After sequence optimisation for the E. coli in vitro system and generating linear templates for wheat germ expression, the success rate of cell-free protein expression reached 93%.ConclusionWe have demonstrated that protein expression in cell-free systems is an appropriate technology for the successful expression of soluble full-length proteins. In our study, wheat germ expression using a two compartment system is the method of choice as it shows high solubility and high protein yield.

Intracellular distribution of rat-liver polyribosomes synthesizing coeruloplasmin.

Three independent approaches (binding of 125I-antibodies to polysomes, cell-free translation of polyadenylated mRNA in a wheat germ system and hybridization of RNA with a specific complementary DNA probe) were used to study the intracellular distribution of polysomes involved in the synthesis of coeruloplasmin as well as of coeruloplasmin mRNA sequences in rat liver. It was shown that only membrane-bound polysomes contain both nascent chains of coeruloplasmin and coeruloplasmin mRNA sequences. The size of coeruloplasmin polysomes is 16-18 monomers/mRNA molecule and their proportion is about 0.4-0.6% of total liver polysomes. The proportion of coeruloplasmin mRNA is 0.0025% of the total polysomal RNA and 0.29% of poly(A)-containing mRNA. These values correspond to coeruloplasmin mRNA concentration about 400-535 molecules per parenchymatous liver cell, 90% of those mRNA molecules were recovered from polysomes while the remaining 10% were from postpolysomal supernatant.

Cell-Free Synthesis of Polypeptides Lacking an Amino-Terminal Methionine by Using a Dicistroviral Intergenic Internal Ribosome Entry Site

An amino-terminal methionine corresponding to a recombinant AUG initiation codon sometimes affects the functions of proteins. To test the performance of translation mediated by a dicistroviral internal ribosome entry site (IRES), which initiates protein synthesis with elongator tRNAs, we optimized the conditions for cell-free translation. Although the IRES is 188 nucleotides long, a further 50 nucleotides of the upstream sequence stabilized translation efficiency. Optimal ion concentrations were affected by the sequences of the constructs. In a wheat-germ system, IRES-mediated translation produced 78 microg/ml of firefly luciferase from the AUG-deleted sequence, suggesting that dicistroviral IRESs will be able to yield polypeptides with a specific N-terminal amino acid other than methionine.

The -4 Phenylalanine Is Required for Substrate Ubiquitination Catalyzed by HECT Ubiquitin Ligases

The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.

Preparation of Functional Ecdysteroid Receptor Proteins (EcR and USP) Using a Wheat Germ Cell-Free Protein Synthesis System

Ecdysone receptor (EcR) and ultraspiracle (USP) of the lepidopteran Chilo suppressalis were expressed using a wheat germ cell-free protein synthesis system, and the functions of the expressed proteins were analyzed. In a gel mobility shift assay, the EcR/USP heterodimer bound to the Drosophila melanogaster hsp27 ecdysone response element. In ligand-receptor binding assays, a potent ecdysteroid, ponasterone A, bound to the EcR/USP complex with a high affinity (KD=2.1 nM). For a series of ecdysteroids and N-tert-butyl-N,N′-dibenzoylhydrazines, the binding activity toward the in vitro expressed EcR/USP complex using a wheat germ system was linearly correlated with that to the endogenous receptor proteins in the cell-free preparation of C. suppressalis integument. These results indicate that functional ecdysteroid receptor proteins were successfully expressed in a wheat germ protein synthesis system. © Pesticide Science Society of Japan

An engineered Escherichia coli tyrosyl-tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system.

Tyrosyl-tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo-L-tyrosine rather than L-tyrosine for the site-specific incorporation of 3-iodo-L-tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-L-tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the L-tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo-L-tyrosine 10-fold more efficiently than L-tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA(Tyr) was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo-l-tyrosine. After the use of the 3-iodotyrosyl-tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of L-tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-L-tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.