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.     

Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium

Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids. Growth in minimal media often results in low yields of cells or expression products. We demonstrate a method in which one labeled amino acid is added to a rich medium. These conditions resulted in high expression (> or =100 mg/L) of a test fusion protein and milligram quantities of the selectively labeled membrane peptide after cyanogen bromide cleavage to release the peptide from the fusion protein. High levels of (15)N incorporation and acceptable levels of cross-labeling into other amino acid residues of the peptide were achieved. Growth in rich media is a simple and convenient alternative to growth in supplemented minimal media and is readily applicable to the expression of proteins selectively labeled with specific amino acids.     

Cell-free synthesis of 15N-labeled proteins for NMR studies

Modern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 N-labeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR spectra recorded of the crude reaction mixture without concentration or chromatographic purification of the protein. We review methodological aspects of cell-free in vitro protein synthesis based on an Escherichia coli cell extract, in particular with regard to the production of 15 N-labeled proteins for analysis by NMR spectroscopy.     

Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.     

In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin.

The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.     

Glyceride-cysteine lipoproteins and secretion by Gram-positive bacteria.

The membrane penicillinases of Bacillus licheniformis and Bacillus cereus are lipoproteins with N-terminal glyceride thioether modification identical to that of the Escherichia coli outer membrane lipoprotein. They are readily labeled with [3H]palmitate present during exponential growth. At the same time, a few other proteins in each organism become labeled and can be detected by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total membrane proteins. We distinguish these proteins from the O-acyl proteolipids by demonstrating the formation of glyceryl cysteine sulfone after performic acid oxidation and hydrolysis of the protein. By this criterion, B. licheniformis and B. cereus contain sets of lipoproteins larger in average molecular weight than that of E. coli. Members of the sets probably are under a variety of physiological controls, as indicated by widely differing relative labeling intensity in different media. The set in B. licheniformis shares with membrane penicillinase a sensitivity to release from protoplasts by mild trypsin treatment, which suggests similar orientation on the outside of the membrane. At least one protein is the membrane-bound partner of an extracellular hydrophilic protein, the pair being related as membrane and exopenicillinases are. We propose that the lipoproteins of gram-positive organisms are the functional equivalent of periplasmic proteins in E. coli and other gram-negative bacteria, prevented from release by anchorage to the membrane rather than by a selectively impermeable outer membrane.     

pGSTag--a versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins.

We report on the construction of a plasmid, pGSTag, that directs the expression in E. coli of a glutathione S-transferase fusion protein that contains a high affinity phosphorylation site by protein kinase-A (PK-A). The fusion protein, following purification from crude bacterial lysates by substrate affinity chromatography, can be labeled in vitro to high specific activity with purified PK-A and 32P-gamma-ATP. Because labeling takes place while the fusion protein is immobilized on a solid support, the unincorporated label and enzyme can be washed away. Using the leucine-zipper domains of cAMP response element binding (CREB) proteins and CCAAT/enhancer binding protein (C/EBP)-like proteins as a model system, we show that the labeled protein, after elution from the affinity resin, can be used as a probe to detect interacting (dimerizing) species in a nitrocellulose-based ligand blot assay. The utility of this system for the creation of labeled protein probes is discussed.     

Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT

Stable isotope-labeled proteotypic peptides are used as surrogate standards for absolute quantification of proteins in proteomics. However, a stable isotope-labeled peptide has to be synthesized, at relatively high cost, for each protein to be quantified. To multiplex protein quantification, we developed a method in which gene design de novo is used to create and express artificial proteins (QconCATs) comprising a concatenation of proteotypic peptides. This permits absolute quantification of multiple proteins in a single experiment. This complete study was constructed to define the nature, sources of error, and statistical behavior of a QconCAT analysis. The QconCAT protein was designed to contain one tryptic peptide from 20 proteins present in the soluble fraction of chicken skeletal muscle. Optimized DNA sequences encoding these peptides were concatenated and inserted into a vector for high level expression in Escherichia coli. The protein was expressed in a minimal medium containing amino acids selectively labeled with stable isotopes, creating an equimolar series of uniformly labeled proteotypic peptides. The labeled QconCAT protein, purified by affinity chromatography and quantified, was added to a homogenized muscle preparation in a known amount prior to proteolytic digestion with trypsin. As anticipated, the QconCAT was completely digested at a rate far higher than the analyte proteins, confirming the applicability of such artificial proteins for multiplexed quantification. The nature of the technical variance was assessed and compared with the biological variance in a complete study. Alternative ionization and mass spectrometric approaches were investigated, particularly LC-ESI-TOF MS and MALDI-TOF MS, for analysis of proteins and tryptic peptides. QconCATs offer a new and efficient approach to precise and simultaneous absolute quantification of multiple proteins, subproteomes, or even entire proteomes.     

Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns

We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.     

Investigating the effects of mutations on protein aggregation in the cell

The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.     

大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体

为进一步探讨大肠杆菌脑微血管内皮细胞侵袭基因ibeB的生物学特性 ,将ibeB基因克隆到pET2 8a(+)载体 ,以E .coliBL2 1 (DE3)为宿主菌 ,经IPTG诱导后 ,通过Ni2 + NTA树脂提纯IbeB蛋白 .SDS PAGE确定纯化蛋白的分子量 ;应用无蛋白酶的体外转录和翻译系统进一步鉴定ibeB基因表达蛋白的分子量 ;通过 [3 5S]Met标记的体内T7表达体系并结合膜蛋白分离技术定位IbeB蛋白在细菌中的亚细胞分布 ;利用细菌侵袭实验分析IbeB蛋白抗体对E .coliK1侵袭人脑微血管内皮细胞的封闭作用 .结果发现 ,ibeB基因的重组蛋白表达纯化产物呈现出 5 0kD和 34kD两种分子量大小 ,5 0kD存在于表达细菌的可溶性部分 ,而 34kD则存在于包涵体中 ;体外翻译实验也显示出较弱的 5 0kD和较浓的 34kD两个蛋白带 ;体内T7表达体系实验显示 34kD的IbeB成熟蛋白定位于E .coli的外膜 ;抗 34kDIbeB蛋白抗体能封闭E .coli对人脑微血管内皮细胞的侵袭 .这些结果提示 ,大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为 5 0kD的外膜蛋白前体 ,该前体可通过分子内剪接形成成熟的 34kDIbeB蛋白     

Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.     

The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination

A recently developed method makes it possible to genetically encode unnatural amino acids with diverse physical, chemical or biological properties in Escherichia coli and yeast. We now show that this technology can be used to efficiently and site-specifically incorporate p-iodo-L-phenylalanine (iodoPhe) into proteins in response to an amber TAG codon. The selective introduction of the anomalously scattering iodine atom into proteins should facilitate single-wavelength anomalous dispersion experiments on in-house X-ray sources. To illustrate this, we generated a Phe153 --> iodoPhe mutant of bacteriophage T4 lysozyme and determined its crystal structure using considerably less data than are needed for the equivalent experiment with cysteine and methionine. The iodoPhe residue, although present in the hydrophobic core of the protein, did not perturb the protein structure in any meaningful way. The ability to selectively introduce this and other heavy atom-containing amino acids into proteins should facilitate the structural study of proteins.     

Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery

Classic in vitro studies show that the Hsp70 chaperone system from Escherichia coli (DnaK-DnaJ-GrpE, the DnaK system) can bind to proteins, prevent aggregation, and promote the correct refolding of chaperone-bound polypeptides into native proteins. However, little is known about how the DnaK system handles proteins that have already aggregated. In this study, glucose-6-phosphate dehydrogenase was used as a model system to generate stable populations of protein aggregates comprising controlled ranges of particle sizes. The DnaK system recognized the glucose-6-phosphate dehydrogenase aggregates as authentic substrates and specifically solubilized and refolded the protein into a native enzyme. The efficiency of disaggregation by the DnaK system was high with small aggregates, but the efficiency decreased as the size of the aggregates increased. High folding efficiency was restored by either excess DnaK or substoichiometric amounts of the chaperone ClpB. We suggest a mechanism whereby the DnaK system can readily solubilize small aggregates and refold them into active proteins. With large aggregates, however, the binding sites for the DnaK system had to be dynamically exposed with excess DnaK or the catalytic action of ClpB and ATP. Disaggregation by the DnaK machinery in the cell can solubilize early aggregates that formed accidentally during chaperone-assisted protein folding or that escaped the protection of "holding" chaperones during stress.     

Protein Phosphorylation in Intact Superior Cervical Ganglion During Regeneration

The incorporation of radioactive phosphate into proteins of both normal and regenerating superior cervical ganglion nerve of the rat is reported. Incorporation studies carried out by in vitro and in vivo methods are compared. In the in vitro method, excised intact ganglia or their homogenates were incubated in the presence of inorganic phosphate or ATP, respectively, under various conditions. Proteins were analyzed by gel electrophoresis followed by autoradiography, in which quantitative but not qualitative differences between regenerating and control cases were apparent. In the in vivo procedure, inorganic phosphate was injected into the living animal 4 h before removal of ganglia. At least fivefold more proteins became labeled in vivo than in vitro, whereas no similarity in the pattern of labeling between the two methods was observed. For example, the most heavily labeled protein in the in vivo method, tentatively identified as microtubule-associated protein-2, was not detected on autoradiograms of proteins labeled by the in vitro method. In this latter method, an 85-kDa species and growth-associated protein-43 were always labeled, and the extent of their phosphorylation was enhanced by the additional presence of phosphatidylserine and Ca2+, a result indicating that these labeled species are substrates of protein kinase C. The in vitro conditions also led to the labeling of proteins identified as alpha- and beta-tubulin. Comparison of the methods suggests that removal of the ganglion interferes with the function of protein phosphorylation systems and that this effect involves elements of the cytoskeleton.     

Identification of a Precursor Pool of Ribosome Protein in Escherichia coli

Antibodies prepared against proteins from 50S ribosomes of Escherichia coli also reacted with the supernatant proteins of a cell-free extract of E. coli which was ribosome-free. A reaction of immunological identity (Ouchterlony tests) was demonstrated for one of these supernatant proteins and one protein found in 50S ribosomes. Isotope experiments involving a shift from (14)C-leucine medium to (12)C-leucine medium showed that these proteins are not formed by breakdown of ribosomes during the preparation of cell-free extracts, but instead represent a pool of ribosome protein which is utilized during growth. In shift experiments from (14)C-leucine to (12)C-leucine medium, the kinetics of disappearance of labeled supernatant ribosome proteins (as measured by reaction with antibody) indicated that half the pool is depleted in 0.1 generation time at 37 C in glucose-salts medium. The pool was also depleted under conditions of amino acid starvation of a "relaxed" strain which accumulated "relaxed" particles. Most, if not all, of the protein present in "relaxed" particles was derived from the pool. The pool represented about 3 to 4% of the total soluble proteins in the ribosome-free supernatant fluid of an E. coli extract.