Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae

Isotope labeling of recombinant proteins is a prerequisite for application of nuclear magnetic resonance spectroscopy (NMR) for the characterization of the three-dimensional structures and dynamics of proteins. Overexpression of isotopically labeled proteins in bacterial or yeast host organisms has several drawbacks. In this work, we tested whether the recently described eukaryotic protein expression system based on the protozoa Leishmania tarentolae could be used for production of amino acid specific (15)N-labeled recombinant proteins. Using synthetic growth medium we were able to express in L. tarentolae and purify to homogeneity (15)N-valine labeled Enchanced Green Fluorescent Protein (EGFP) with the final yield of 5.7 mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identification of all 18 valine residues of the protein in the HSQC spectrum. Our results demonstrate the suitability of the L. tarentolae expression system for production of isotopically labeled proteins.     

Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75 kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17 kDa), at 12 to 24 h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24 h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins. Received October 9, 2001 Accepted January 18, 2002     

Stable isotope labeling of protein by Kluyveromyces lactis for NMR study

Stable isotope labeling for proteins of interest is an important technique in structural analyses of proteins by NMR spectroscopy. Escherichia coli is one of the most useful protein expression systems for stable isotope labeling because of its high-level protein expression and low costs for isotope-labeling. However, for the expression of proteins with numerous disulfide-bonds and/or post-translational modifications, E. coli systems are not necessarily appropriate. Instead, eukaryotic cells, such as yeast Pichia pastoris, have great potential for successful production of these proteins. The hemiascomycete yeast Kluyveromyces lactis is superior to the methylotrophic yeast P. pastoris in some respects: simple and rapid transformation, good reproducibility of protein expression induction and easy scale-up of culture. In the present study, we established a protein expression system using K. lactis, which enabled the preparation of labeled proteins using glucose and ammonium chloride as a stable isotope source.     

Automated structure determination of proteins with the SAIL-FLYA NMR method

The labeling of proteins with stable isotopes enhances the NMR method for the determination of 3D protein structures in solution. Stereo-array isotope labeling (SAIL) provides an optimal stereospecific and regiospecific pattern of stable isotopes that yields sharpened lines, spectral simplification without loss of information, and the ability to collect rapidly and evaluate fully automatically the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as those that can be analyzed using conventional methods. Here, we describe a protocol for the preparation of SAIL proteins by cell-free methods, including the preparation of S30 extract and their automated structure analysis using the FLYA algorithm and the program CYANA. Once efficient cell-free expression of the unlabeled or uniformly labeled target protein has been achieved, the NMR sample preparation of a SAIL protein can be accomplished in 3 d. A fully automated FLYA structure calculation can be completed in 1 d on a powerful computer system.     

Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium

Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.     

Forceful large-scale expression of "problematic" membrane proteins

We developed an Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches. The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthesized protein, and selection of a compatible expression host. This enabled us to enhance the expression level of a toxic membrane protein 30-50 times compared with expression in the native state and to obtain 3-5mg of a highly purified functionally active protein per liter of culture. We describe the method for the amplified expression of membrane proteins, using the Pseudomonas aeruginosa multidrug resistance protein, MexY, as an example. The amplified MexY was correctly folded in the cytoplasmic membrane of the E. coli without forming inclusion bodies. This method can be applicable to the large-scale expression of the other problematic membrane proteins that are otherwise extremely difficult to overproduce.     

A genetically encodable microtag for chemo-enzymatic derivatization and purification of recombinant proteins

Efficient separation of recombinant polypeptides from proteins of the expression host and their subsequent derivatisation with functional chemical groups is essential for the success of many biological applications. Numerous tag systems have been developed to facilitate the purification procedure but only limited progress has been made in development of generic methods for targeted modification of proteins with functional groups. In this work, we present a novel 6 amino acid long C-terminal protein tag that can be selectively modified with functionalized derivatives of farnesyl isoprenoids by protein farnesyltransferase. The reaction could be performed in complex protein mixtures without detectable unspecific labeling. We demonstrate that this modification can be used to purify the target protein by over 800-fold in a single purification step using phase partitioning. Moreover, we show that the fluorescent group could be used to monitor the interaction of the derivatized proteins with other polypeptides.     

Recombinant baculovirus-based multiple protein expression platform for Drosophila S2 cell culture

A platform for selective and controllable expression of multiple foreign protein types was developed in insect cell culture. Based on the fact that baculovirus cannot replicate in nonpermissive Drosophila melanogaster Schneider line 2 (S2) cells, S2 cells that stably express human erythropoietin (hEPO) under the control of the S2-derived inducible metallothionein (MT) promoter were infected with three types of recombinant baculoviruses, each of which expressed a different fluorescent protein gene under the control of MT promoter. Addition of copper sulfate as an inducer to infected, stably transfected S2 cells resulted in simultaneous expression of hEPO and three fluorescent proteins. Expression profiles and levels of the three induced fluorescent proteins were similar in all single infected cells. Importantly, expression profiles and levels of hEPO were similar in both non-infected and infected cells, indicating that baculovirus expressed recombinant proteins do not adversely affect expression of host cell recombinant proteins. Expressions of the three fluorescent proteins were able to be selectively regulated by altering combination ratios of the three types of recombinant baculoviruses. Collectively, these data indicate that the baculovirus/stably transfected S2 cell system can be successfully used to express multiple foreign proteins in a controlled and selective manner without the burden of additional selection markers. Such a system would be expected to be attractive as a multiple protein expression platform for engineering metabolic or glycosylation pathways.     

Quantitative proteomes and in vivo secretomes of progressive and regressive UV-induced fibrosarcoma tumor cells: mimicking tumor microenvironment using a dermis-based cell-trapped system linked to tissue chamber

The alterations of tumor proteome and/or in vivo secretome created by host-tumor cell interaction may be crucial factors for tumors to undergo progression or regression in a host system. Two UV-induced fibrosarcoma tumor cell lines (UV-2237 progressive cells and UV-2240 regressive cells) were used as models to address this issue. Hundreds of proteins including in vivo secretome have been identified and quantified via an isotope-coded protein label (ICPL) in conjunction with high-throughput NanoLC-LTQ MS analysis. A newly designed technology using a dermis-based cell-trapped system was employed to encapsulate and grow 3-D tumor cells. A tissue chamber inserted with a tumor cell-trapped dermis was implanted into mice to mimic the tumor microenvironment. The in vivo secretome created by host-tumor interaction was characterized from samples collected from tissue chamber fluids via ICPL labeling mass spectrometric analysis. Twenty-five proteins including 14-3-3 proteins, heat shock proteins, profilin-1, and a fragment of complement C3 with differential expression in proteomes of UV-2237 and UV-2240 cells were revealed. Three secreted proteins including myeloperoxidase, alpha-2-macroglobulin, and a vitamin D-binding protein have different abundances in the in vivo secretome in response to UV-2237 and UV-2240 cells. Differential tumor proteomes and in vivo secretome were thus accentuated as potential therapeutic targets to control tumor growth.     

Mass spectrometry reveals specific and global molecular transformations during viral infection

Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.     

"Cystope tagging" for labeling and detection of recombinant protein expression

A labeling and detection method, based on the addition of a single cysteine residue at the C terminus of a recombinant protein and the subsequent sulfhydryl-specific Michael addition to the double bond of maleimide and its derivatives, was developed. The method was named "cystope tagging." Sorbit dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase family of proteins that contains three inherent cysteines, was used as a model recombinant protein. By labeling with fluorescein-maleimide, it was demonstrated that only the single accessory cysteine is accessible under nonreducing conditions. After the addition of beta-mercaptoethanol, the inherent cysteines of SDH were also detectable by coupling to fluorescein-maleimide. The data were obtained using Autodisplay, an efficient surface expression system in Escherichia coli, but the method presented in this article represents a rather general solution for analyzing the expression of recombinant proteins, irrespective of the expression system used. The authors conclude that cystope tagging is an interesting alternative to other tagging methods applied in recombinant protein techniques.     

Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS

Targeted quantification of proteins is a daily task in biological research but often relies on techniques such as western blotting that are only barely quantitative. Here we present a broadly applicable workflow for protein quantification from unpurified whole-cell extracts that can be completed in less than 3 d. Without prefractionation or affinity enrichment, a whole-cell extract is trypsin-digested in an acetonitrile-containing ammonium carbonate buffer and high-molecular-weight compounds are removed by filtration. A normalization strategy, which involves endogenous reference proteins, facilitates the determination of relative changes in protein expression without requiring isotope labeling or standard addition. On a triple-quadrupole mass spectrometer, we demonstrate standard-free quantification of yeast proteins present over five orders of magnitude and present at ≥500 copies per cell. Liquid chromatography/multiple reaction monitoring (LC-MRM)-based proteomics is therefore a next-generation alternative to western blotting, as it allows simultaneous and reliable quantification of multiple endogenous proteins without the need for enrichment, isotope labeling or use of antibodies.     

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.     

Intein-based biosynthetic incorporation of unlabeled protein tags into isotopically labeled proteins for NMR studies

Segmental isotopic labeling of proteins using protein ligation is a recently established in vitro method for incorporating isotopes into one domain or region of a protein to reduce the complexity of NMR spectra, thereby facilitating the NMR analysis of larger proteins. Here we demonstrate that segmental isotopic labeling of proteins can be conveniently achieved in Escherichia coli using intein-based protein ligation. Our method is based on a dual expression system that allows sequential expression of two precursor fragments in media enriched with different isotopes. Using this in vivo approach, unlabeled protein tags can be incorporated into isotopically labeled target proteins to improve protein stability and solubility for study by solution NMR spectroscopy.     

Cell-free complements in vivo expression of the E. coli membrane proteome

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.     

A novel antibody microarray format using non-covalent antibody immobilization with chemiluminescent detection

To date, protein and antibody microarrays have been used in reverse-phase and sandwich-based methods in order to detect known proteins such as biomarkers in samples. Our group developed "libraries" of antibodies against unknown proteins, referred to as mKIAA proteins, and we attempted to discover candidate novel biomarkers by protein expression profiling.To profile mKIAA protein expression using these antibodies, we established an antibody microarray system using chemiluminescent detection. A number of techniques for protein-antibody microarrays have been reported; however, no entirely suitable protocol for crude protein samples has been established. To address this issue, we immobilized purified antibodies on hydrophilic surface polymer slides (Maxisorp, Nunc). Although our system is based on the direct labeling of crude protein samples, we achieved sufficient sensitivity (detection limit: 50 pg mL(-1)) and low backgrounds. This sensitivity is on a level with the sandwich immunoassay-based antibody array system. Using our protocol, we developed an antibody microarray spotted with 960 anti-mKIAA antibodies (total: 3888 spots for quadruplicate assessments), and we carried out protein expression profiling of mKIAA proteins. In this study, we generated an expression profile of 960 mKIAA proteins and compared the present results with those obtained via cDNA microarray.     

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote <Emphasis Type="Italic">Pichia pastoris</Emphasis>

¹³C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific ¹³C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient ¹³C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.     

Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression saccharomyces cerevisiae strains: the comparison of two quantitative methods

The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches was used to identify relative changes in the protein expression levels between the strains. A total of 2388 proteins were relatively quantified, and more than 350 proteins were found to have significantly different expression levels between the two strains of comparison when using the stable isotope labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found, the major reason behind the discrepancy was the lack of reproducible sampling for proteins with low spectral counts. The functional categorization of the relative protein expression differences that occur in Snf1-deficient strains uncovers a wide range of biological processes regulated by this important cellular kinase.