A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

One‐step refolding and purification of recombinant human tumor necrosis factor‐α (rhTNF‐α) using ion‐exchange chromatography

Protein refolding is a key step for the production of recombinant proteins, especially at large scales, and usually their yields are very low. Chromatographic‐based protein refolding techniques have proven to be superior to conventional dilution refolding methods. High refolding yield can be achieved using these methods compared with dilution refolding of proteins. In this work, recombinant human tumor necrosis factor‐α (rhTNF‐α) from inclusion bodies expressed in Escherichia coli was renatured with simultaneous purification by ion exchange chromatography with a DEAE Sepharose FF column. Several chromatographic parameters influencing the refolding yield of the denatured/reduced rhTNF‐α, such as the urea concentration, pH value and concentration ratio of glutathione/oxidized glutathione in the mobile phase, were investigated in detail. Under optimal conditions, rhTNF‐α can be renatured and purified simultaneously within 30 min by one step. Specific bioactivity of 2.18 × 10⁸ IU/mg, purity of 95.2% and mass recovery of 76.8% of refolded rhTNF‐α were achieved. Compared with the usual dilution method, the ion exchange chromatography method developed here is simple and more effective for rhTNF‐α refolding in terms of specific bioactivity and mass recovery. Copyright © 2014 John Wiley & Sons, Ltd.     

Glutathionylated γG and γA subunits of hemoglobin F: a novel post‐translational modification found in extremely premature infants by LC‐MS and nanoLC‐MS/MS

Oxidative stress plays an important role in the development of various disease processes and is a putative mechanism in the development of bronchopulmonary dysplasia, the most common complication of extreme preterm birth. Glutathione, a major endogenous antioxidant and redox buffer, also mediates cellular functions through protein thiolation. We sought to determine if post‐translational thiol modification of hemoglobin F occurs in neonates by examining erythrocyte samples obtained during the first month of life from premature infants, born at 23 0/7 – 28 6/7 weeks gestational age, who were enrolled at our center in the Prematurity and Respiratory Outcomes Program (PROP). Using liquid chromatography‐mass spectrometry (LC‐MS), we report the novel finding of in vivo and in vitro glutathionylation of γG and γA subunits of Hgb F. Through tandem mass spectrometry (nanoLC‐MS/MS), we confirmed the adduction site as the Cys‐γ94 residue and through high‐resolution mass spectrometry determined that the modification occurs in both γ subunits. We also identified glutathionylation of the β subunit of Hgb A in our patient samples; we did not find modified α subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of γG and γA of Hgb F occurs in premature infants. Additional studies of this post‐translational modification are needed to determine its physiologic impact on Hgb F function and if sG‐Hgb is a biomarker for clinical morbidities associated with oxidative stress in premature infants. Copyright © 2014 John Wiley & Sons, Ltd.     

Telechelic,Antimicrobial Hydrophilic Polycations with Two Modes of Action

Telechelic antimicrobial poly(2‐oxazoline)s with quaternary ammonium (quat) end groups are shown to be potent antimicrobial polymers against Gram‐positive bacterial strains. In this study, the activity against the Gram‐negative bacterium Escherichia coli is additionally implemented by hydrolyzing the poly(2‐methyl‐2‐oxazoline) with two quart end groups to poly(ethylene imine) (PEI). The resulting telechelic polycations are active against Staphylococcus aureus and E. coli. The contribution of the PEI backbone is determined by measuring the antimicrobial activity in the presence of calcium ions. The influence of PEI on the overall activity strongly depends on the molecular weight and increases with higher mass. The PEI dominates the activity against E. coli at lower masses than against S. aureus. The quart end groups require an alkyl substituent of dodecyl or longer to dominate the antimicrobial activity. Additionally, PEI and quart end groups act synergistically.     

Defining topological features of membrane proteins by nanoelectrospray ionisation mass spectrometry

The D‐galactose‐H⁺ symport protein, GalP, of Escherichia coli is the bacterial homologue of the human glucose transport protein, GLUT1. Here we demonstrate that mass spectrometry can be used to map modification by covalently bound reagents, and also to detect structural changes in the GalP protein that occur upon substrate binding. The small thiol‐group‐specific reagent N‐ethylmaleimide (NEM) was used to modify the cysteine residues in GalP(His)₆ both alone and in the presence of D‐glucose, a known substrate. Employing a mixture of proteolysis and thermal degradation methods, the three cysteine residues were found to undergo sequential reactions with NEM, with Cys374 being modified first, followed by Cys389 and finally Cys19, thus indicating their different accessibilities within the three‐dimensional structure of the protein. Prior binding of the substrate D‐glucose to the protein protected Cys19 and Cys374 against NEM modification, but not Cys389. Cys374 had been expected to be shielded by D‐glucose binding while Cys389 had been expected to be unaffected, consistent with their proposed respective locations in the vicinity of, and distant from, the sugar binding site. However, the inaccessibility of Cys19 was unexpected and suggests a structural change in the protein promoted by D‐glucose binding which changes the proximity of Cys19 with respect to the D‐glucose‐binding site. Copyright © 2010 John Wiley & Sons, Ltd.     

Purification and Refolding of Cyclodextrin Glycosyltransferase Expressed Escherichia coli

Recombinant DNA technology and protein engineering are currently utilized in the cost-effective production of pharmaceutical and industrial proteins with native conformation. Escherichia coli retains its dominant position as the first choice of host for speed, simplicity and well-established production protocols. However, protein production using recombinant E. coli occasionally encounters complex purification and refolding steps. This paper introduces an efficient scheme for purification andin vitro refolding of industrially important proteins including cyclodextrin glycosyltransferase (CGTase) expressed in recombinant E. coli employing a polycationic amino acid fusion system. Fusion of polycationic amino acids to CGTase allowed purification and refolding of CGTase to be simple and efficient. A novel CGTase production strategy will be discussed by considering recent progress in protein purification and refolding techniques.     

Isolation,Purification, and Properties of a Novel Small Heat Shock Protein from the Hyperthermophile <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

The isolation, purification, and properties of a putative small heat shock protein (sHsp), named SsHSP14.1, from the hyperthermophilic archaeon Sulfolobus solfataricus have been investigated. The sHsp was successfully expressed and purified from Escherichia coli. In vivo chaperone function of SsHSP14.1 for preventing aggregation of proteins during heating was investigated. It was found that recombinant SsHSP14.1 with a molecular mass of 17.8 kDa prevented E. coli proteins from aggregating in vivo at 50 °C. This result suggested that SsHSP14.1 confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures. In vitro, the purified SsHSP14.1 protein was able to prevent Candida antarctica lipase B from aggregation for up to 60 min at 80 °C. Moreover, the SsHSP14.1 enhanced thermostability of bromelain extending its half-life at 55 °C by 67%.     

Cloning and Expression of Chitinase A from Serratia Marcescens for Large‐Scale Preparation of N,N‐Diacetyl Chitobiose

The gene of Serratia marcescens chitinase A (chiA) was cloned by PCR. The complete gene was constructed into a pRSET vector and expressed in Escherichia coli. The recombinant enzyme was purified to > 90% homogeneity by hydrophobic interaction chromatography followed by ion‐exchange separation. Measured with an electrospray‐ionization mass spectrometer, the molecular mass of the protein was 58,607 Da, consistent with a theoretical calculation of the deduced protein without the signal peptide. The recombinant enzyme was characterized and tested for the preparation of chitobiose. In general, the recombinant Chtinase A exhibited an exo‐type catalytic activity toward colloidal chitin and released both N‐acetylglucosamine and N,N‐diacetyl chitobiose as products. After extensive testing, we produced N,N‐diacetyl chitobiose as the predominant product when the enzymatic reaction was performed in sodium acetate buffer at pH 5.5; under such conditions, an enzymatic process is established for the production of the disaccharide on a 100‐g scale.     

A Rigidoporus Vinctus Alcohol Dehydrogenase and Its Characterization

Alcohol dehydrogenases (ADHs; E.C. 1.1.1.1) are widely distributed enzymes found in many microorganisms. ADHs are oxidoreductases that catalyze the NAD(P)⁺‐dependent conversion of alcohols to aldehydes or ketones as well as the reverse reaction. The ADH cloned from Rigidoporus vinctus (RvADH) was 1035 bp that encodes a protein of 344 amino acid residues with calculated molecular mass of 38.39 kDa. This ADH is belonging to the medium‐chain family (medium‐chain dehydrogenase/reductase (MDR) and has the highly conserved GXXGXXG sequence found in the MDR family which found as the coenzyme‐binding pocket. To characterize the ADH protein, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged ADH was overexpressed and purified by Ni²⁺‐nitrilotriacetic acid Sepharose. The purified enzyme showed one band on 12 % sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Michaelis constant (K_M) value of the recombinant enzyme for ethanol was 0.79 mM. In substrates specificity analysis showed that RvADH had great oxidative activity toward primary alcohols. However, the less activtiy toward secondary alcohols and alcohol derivatives were compared with ethanol. Regarding the reductase activity showed low or even no activity to aldehydes and ketone.     

Covalent selection of the thiol proteome on activated thiol sepharose: A robust tool for redox proteomics

Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (-SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of -SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching -SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. -SH-containing proteins account for 2.52% (E. coli), 1.4% (T. harzianum) and 1.4% (M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M_r thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for -SH-containing proteins in redox proteomics.     

Peptide peak intensities enhanced by cysteine modifiers and MALDI TOF MS

Two cysteine‐specific modifiers we reported previously, N‐ethyl maleimide (NEM) and iodoacetanilide (IAA), have been applied to the labeling of cysteine residues of peptides for the purpose of examining the enhancement of ionization efficiencies in combination with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI TOF MS). The peak intensities of the peptides as a result of modification with these modifiers were compared with the peak intensities of peptides modified with a commercially available cysteine‐specific modifier, iodoacetamide (IA). Our experiments show significant enhancement in the peak intensities of three cysteine‐containing synthetic peptides modified with IAA compared to those modified with IA. The results showed a 4.5–6‐fold increase as a result of modification with IAA compared to modification with IA. Furthermore, it was found that IAA modification also significantly enhanced the peak intensities of many peptides of a commercially available proteins, bovine serum albumin (BSA), compared to those modified with IA. This significant enhancement helped identify a greater number of peptides of these proteins, leading to a higher sequence coverage with greater confidence scores in identification of proteins with the use of IAA. Copyright © 2012 John Wiley & Sons, Ltd.     

Phosphorothioate Modification of mRNA Accelerates the Rate of Translation Initiation to Provide More Efficient Protein Synthesis

Messenger RNAs (mRNAs) with phosphorothioate modification (PS‐mRNA) to the phosphate site of A, G, C, and U with all 16 possible combinations were prepared, and the translation reaction was evaluated using an E. coli cell‐free translation system. Protein synthesis from PS‐mRNA increased in 12 of 15 patterns when compared with that of unmodified mRNA. The protein yield increased 22‐fold when the phosphorothioate modification at A/C sites was introduced into the region from the 5′‐end to the initiation codon. Single‐turnover analysis of PS‐mRNA translation showed that phosphorothioate modification increases the number of translating ribosomes, thus suggesting that the rate of translation initiation (rate of ribosome complex formation) is positively affected by the modification. The method provides a new strategy for improving translation by using non‐natural mRNA.     

Characterization of dehydroascorbate‐mediated modification of glutaredoxin by mass spectrometry

Ascorbate is as a potent antioxidant in vivo protecting the organism against oxidative stress. In this process, ascorbate is oxidized in two steps to dehydroascorbate (DHA), which if not efficiently reduced back to ascorbate decomposes irreversibly to a complex mixture of products. We demonstrate that a component of this mixture specifically reacts with the thiol group of cysteine residues at physiological pH to give a protein adduct involving the addition of a 5‐carbon fragment of DHA (+112 Da). Incubations of glutaredoxin‐1 expressed in Escherichia coli and dehydroascorbate revealed abundant adducts of +112, +224 and +336 Da due to the addition of one, two and three conjugation products of DHA, respectively. ESI–MS of carbamidomethylated glutaredoxin‐1 before incubation with DHA, deuterium exchange together with tandem mass spectrometry analysis and LC–ESIMS/MS of modified peptides confirmed structure and sites of modification in the protein. Modification of protein thiols by a DHA‐derived product can be involved in oxidative stress‐mediated cellular toxicity. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparation of glycopolymer‐substituted gold nanoparticles and their molecular recognition

Glycopolymer‐substituted gold nanoparticles were prepared via living radical polymerization with a reversible addition‐fragmentation chain transfer (RAFT) reagent. Polyacrylamide derivatives with α‐mannose (α‐Man) and N‐acetyl‐β‐glucosamine (β‐GlcNAc) were synthesized and hydrogenated to obtain thiol‐terminated polymer. The thiol‐terminated glycopolymers were mixed with gold nanoparticles to yield the polymer substituted gold nanoparticles with various diameters, which aggregated on addition of saccharide‐recognition proteins (lectins). The aggregation properties were analyzed using transmission electron microscopy and UV spectra. Molecular recognition was studied with E. coli, which induced aggregation of the nanoparticles at the cell periphery. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1412–1421, 2009     

Cloning and Characterization of a Sucrose Isomerase from <Emphasis Type="Italic">Erwinia rhapontici</Emphasis> NX-5 for Isomaltulose Hyperproduction

The sucrose isomerase (SIase) gene from an efficient strain of Erwinia rhapontici NX-5 for isomaltulose hyperproduction was cloned and overexpressed in Escherichia coli. Protein sequence alignment revealed that SIase was a member of the glycoside hydrolase 13 family. The molecular mass of the purified recombinant protein was estimated at 66 kDa by SDS-PAGE. The SIase had an optimal pH and temperature of 5.0 and 30 °C, respectively, with a K _m of 257 mmol/l and V _max of 48.09 μmol/l/s for sucrose. To the best of our knowledge, the recombinant SIase has the most acidic optimum pH for isomaltulose synthesis. When the recombinant E. coli (pET22b- palI) cells were used for isomaltulose synthesis, almost complete conversion of sucrose (550 g/l solution) to isomaltulose was achieved in 1.5 h with high isomaltulose yields (87%). The immobilized E. coli cells remained stable for more than 30 days in a “batch”-type enzyme reactor. This indicated that the recombinant SIase could continuously and efficiently produce isomaltulose.     

Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry

We report covalent attachment via a thiol ester linkage of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) to cysteine-containing protein biomarkers from bacterial cell lysates of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry when using SA as the matrix. Evidence to support this conclusion is the appearance of additional peaks in the MS spectra when using SA, which are absent when using α-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) ∼208 greater to the m/z of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight (TOF-TOF) mass spectrometer and top-down proteomics. Three protein biomarkers, HdeA, HdeB, and homeobox or YbgS (each containing two cysteine residues) were identified as having reactivity to SA. Non-cysteine-containing protein biomarkers showed no evidence of reactivity to SA. MS ions and MS/MS fragment ions were consistent with covalent attachment of SA via a thiol ester linkage to the side-chain of cysteine residues. MS/MS of a protein biomarker ion with a covalently attached SA revealed fragment ion peaks suggesting dissociative loss SA. We propose dissociative loss of SA is facilitated by a pentacyclic transition-state followed by proton abstraction of the β-hydrogen of the bound SA by a sulfur lone pair followed by dissociative loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal. The apparent reactivity of SA to cysteine/disulfide-containing proteins may complicate identification of such proteins, however the apparent differential reactivity of SA and HCCA toward cysteine/disulfide-containing proteins may be exploited for identification of unknown cysteine-containing proteins.     

Thiol‐disulfide redox equilibria of glutathione metaboloma compounds investigated by tandem mass spectrometry

The thiol group of cysteine plays a pivotal role in structural and functional biology. We use mass spectrometry to study glutathione‐related homo‐ and heterodimeric disulfides, aiming at understanding the factors affecting the redox potentials of different disulfide/thiol pairs. Several electrospray ionization (ESI)‐protonated disulfides of cysteamine, cysteine, penicillamine, N‐acetylcysteine, N‐acetylpenicillamine, γGluCySH, HSCyGly, and glutathione were analyzed on a triple quadrupole instrument to measure their energy‐resolved tandem mass spectra. Fission of the disulfide bond yields RSH*H⁺ and RS⁺ ions. The logarithm of the intensity ratio of the RS⁺/RSH*H⁺ fragments in homodimeric disulfides is proportional to the normal reduction potential of their RSSR/RSH pairs determined by nuclear magnetic resonance (NMR) in solution, the more reducing ones yielding the higher ratios. Also in some R₁S‐SR₂ disulfides, the ratio of the intensities of the RSH + H⁺ and RS⁺ ions of each participating thiol shows a linear relationship with the Nernst equation potential difference of the corresponding redox pairs. This behavior allows us to measure the redox potentials of some disulfide/thiol pairs by using different thiol‐reducing probes of known oxidoreductive potential as reference. To assist understanding of the fission mechanism of the disulfide bond, the fragments tentatively identified as ‘sulfenium’ were themselves fragmented; accurate mass measurement of the resulting second‐generation fragments demonstrated a loss of thioformaldehyde, thus supporting the assigned structure of this elusive intermediate of the oxidative stress pathway. Understanding this fragmentation process allows us to employ this technique with larger molecules to measure by mass spectrometry the micro‐redox properties of different disulfide bonds in peptides with catalytic and signaling biological activity. Copyright © 2008 John Wiley & Sons, Ltd.     

Engineering of Cysteine Residues Leads to Improved Production of a Human Dipeptidase Enzyme in <Emphasis Type="Italic">E. coli</Emphasis>

Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human β-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem β-lactam antibiotics and can cleave dipeptides containing amino acids in both d- and l-configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.     

Cytokines Expression Induced by <Emphasis Type="Italic">Ganoderma sinensis</Emphasis> Fungal Immunomodulatory Proteins (FIP-gsi) in Mouse Spleen Cells

Ganoderma sinensis fungal immunomodulatory protein (FIP-gsi) was a new member of FIPs family. Based on the cloning of FIP-gsi gene from G. sinensis, this paper reported that FIP-gsi gene was expressed in Escherichia coli expression system. Then, the recombinant proteins were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Finally, the immunomodulatory activity was examined by inducing cytokine genes expression. The results showed that the recombinant FIP-gsi protein could be expressed in E. coli and got the yield of about 25% of the soluble form in the total soluble protein. The FIP-gsi protein was composed of 111 amino acids, and the sequence of homologous rate was 88.6% with FIP-glu (LZ-8). Furthermore, it could enhance the levels of interleukin (IL)-2, IL-3, IL-4, interferon gamma, tumor necrosis factor alpha, and IL-2 receptor (IL-2R) in mouse spleen cells.