Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.     

Enhanced Thermotolerance of <Emphasis Type="Italic">E. coli</Emphasis> by Expressed OsHsp90 from Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A gene encoding the rice (Oryza sativa L.) 90-kDa heat shock protein (OsHsp90) was introduced into Escherichia coli using the pGEX-6p-3 expression vector with a glutathione-S-transferase (GST) tag to analyze the possible function of this protein under heat stress for the first time. We compared the survivability of E. coli (BL21) cells transformed with a recombinant plasmid containing GST-OsHsp90 fusion protein with control E. coli cells transformed with the plasmid containing GST and the wild type BL21 under heat shock after isopropyl β-d-thiogalactopyranoside induction. Cells expressing GST-OsHsp90 demonstrated thermotolerance at 42, 50, and 70°C, treatments that were more harmful to cells expressing GST and the wild type. Further studies were carried out to analyze the heat-induced characteristics of OsHsp90 at 42, 50, and 70°C in vitro. When cell lysates from E. coli transformants were heated at these heat stresses, expressed GST-OsHsp90 prevented the denaturation of bacterial proteins treated with 42°C heat shocks, and partially prevented that of proteins treated at 50 and 70°C; meanwhile, cells expressing GST-OsHsp90 withstood the duration at 50°C. These results indicate that OsHsp90 functioned as a chaperone, binding to a subset of substrates, and maintained E. coli growth well at high temperatures.     

Gene Cloning of Endoglucanase Cel5A from Cellulose-Degrading <Emphasis Type="Italic">Paenibacillus xylanilyticus</Emphasis> KJ-03 and Purification and Characterization of the Recombinant Enzyme

The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.     

Identification of cold-inducible inner membrane proteins of the psychrotrophic bacterium, Shewanella livingstonensis Ac10, by proteomic analysis

Shewanella livingstonensis Ac10 is a psychrotrophic Gram-negative bacterium that grows at temperatures close to 0°C. Previous proteomic studies of this bacterium identified cold-inducible soluble proteins and outer membrane proteins that could possibly be involved in its cold adaptation (Kawamoto et al. in Extremophiles 11:819–826, 2007). In this study, we established a method for separating the inner and outer membranes by sucrose density gradient ultracentrifugation and performed proteomic studies of the inner membrane fraction. The cells were grown at temperatures of 4 and 18°C, and phospholipid-enriched inner membrane fractions were obtained. Two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting analysis of the proteins identified 14 cold-inducible proteins (more than a 2-fold increase at 4°C). Six of these proteins were predicted to be inner membrane proteins. Two predicted periplasmic proteins, 5 predicted cytoplasmic proteins, and 1 predicted outer membrane protein were also found in the inner membrane fraction, suggesting their association with the inner membrane proteins and/or lipids. These cold-inducible proteins included proteins that are presumed to be involved in chemotaxis (AtoS and PspA), membrane protein biogenesis (DegP, SurA, and FtsY), and morphogenesis (MreB). These findings provide a basis for further studies on the cold-adaptation mechanism of this bacterium.     

Proteome response of Escherichia coli fed-batch culture to temperature downshift

During fed-batch cultivation of Escherichia coli K-12, the proteomic response to a temperature downshift from 37 to 20°C was quantitatively monitored and analyzed by using two-dimensional electrophoresis. When the temperature of exponentially growing E. coli K-12 culture was downshifted to 20°C, the synthesis level of 57 intracellular proteins showed significant changes for a prolonged period of time, compared to the fed-batch culture controlled at 37°C. Thus, these proteins are regarded as important stress proteins responsive to cold shock, which were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and identified using the E. coli SWISS-2DPAGE database. Most of the identified proteins were shown to be involved in energy metabolism, several cellular molecule biosynthetic pathways and catabolism, cell processes, flagellar biosynthesis and motility, and protein translation and folding. The systematic approach to the monitoring of proteomic responses and the detailed analysis results reported in this article would be useful in understanding the metabolic adaptation to lowered culture temperature and designing efficient fermentation strategies for the production of recombinant proteins and metabolites using E. coli strains.     

Characterization of a thermostable cyclodextrin glucanotransferase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis> DSM3638

A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase.     

A β-barrel outer membrane protein facilitates cellular uptake of polychlorophenols in <Emphasis Type="Italic">Cupriavidus necator</Emphasis>

The tcpRXABCYD operon of Cupriavidus necator JMP134 is involved in the degradation of 2,4,6-trichlorophenol (TCP). All of the gene products except TcpY have assigned functions in TCP metabolism. Sequence comparison identified TcpY as a member of COG4313, a group of hypothetical proteins. TcpY has a signal peptide, indicating it is a membrane or secreted protein. Secondary structure and topology analysis indicated TcpY as a β-barrel outer membrane protein, similar to the Escherichia coli outer membrane protein FadL that transports hydrophobic long-chain fatty acids. Constitutive expression of tcpY in two C. necator strains rendered the cells more sensitive to TCP and other polychlorophenols. Further, C. necator JMP134 expressing cloned tcpY transported more TCP into the cell than a control with the cloning vector. Thus, TcpY is an outer membrane protein that facilitates the passing of polychlorophenols across the outer membrane of C. necator. Similarly, other COG4313 proteins are possibly outer membrane transporters of hydrophobic aromatic compounds.     

Production of recombinant antibody fragments in <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Background  

Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.     

Expression and function of two chaperone proteins,AtGroEL and AtGroES,from Acidithiobacillus ferrooxidans ATCC 23270

Two molecular chaperone genes encoding the Acidithiobacillus ferrooxidans Hsp60 (AtGroEL) and Hsp10 (AtGroES), respectively were introduced into Escherichia coli using the pLM1 expression vector. Then the AtGroEL and AtGroES proteins were overexpressed successfully in Escherichia coli BL21 (DE3), and purified by one-step immobilized metal affinity chromatography. The ATPase assay showed that the proteins were in active form, and the ATPase activity of AtGroEL was temperature dependent with an optimal temperature of 50°C, but the co-chaperonin AtGroES inhibited the ATPase activity of AtGroEL. The chaperonin function of the recombinant proteins was examined using three different protein substrates in vitro, and the results showed that AtGroEL/AtGroES chaperone system could facilitate the refolding of the thermodenatured rusticyanin and recover the activity of thermodenatured ArsH protein. In addition, it could improve the thermal stability of xylanase. Molecular modelling for AtGroEL protein revealed that residues of Tyr199, Ser201, Tyr203, Phe204, Leu234, Leu237, Leu259, Val263 and Val264 were necessary for binding the denatured polypeptides.     

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins

The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor β chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20°C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10⁹ cfu ml⁻¹) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10⁴ cfu ml⁻¹ at 4°C, and no viable cells were detected at 20°C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l⁻¹ for superantigens and 2 mg l⁻¹ for T-cell receptor β chain. These results contribute to the development of methods for microbial cell preservation under field conditions. Martín F. Desimone and Mauricio C. De Marzi contributed equally to this work     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43°C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20°C, but in a liquid crystalline state when cells were grown at 37 and 43°C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Modification of a ferric enterobactin receptor protein from the outer membrane of Escherichia coli.

Modification of a ferric enterobactin receptor protein of Escherichia coli was observed upon incubation of either whole membranes or Triton X-100 solubilized outer membrane at 37°C. The modification was characterized by a change in mobility of the receptor band on SDS polyacrylamide gel electrophoresis and by a decreased binding capacity for ferric enterobactin. The rate of modification was affected by temperature and trypsin inhibitor, benzamidine. Ferric enterobactin inhibited the reaction in whole membrane. The modification affected the limited chymotrypsin digestion pattern of the receptor. The activity may represent a specific modification of the receptor, one possibly mediated by a membran-associated enzyme.     

Enhancement of glutathione production with a tripeptidase-deficient recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutathione (GSH) degradation exists in the enzymatic synthesis of GSH by Escherichia coli, however, its degradation pathway is not very clear. This paper examines the key enzymes responding to GSH degradation in E. coli with the purpose of improving GSH production. The enzymes that are probably associated with GSH degradation were investigated by disrupting their genes. The results suggested that γ-glutamyltranspeptidase (GGT) and tripeptidase (PepT) were the key enzymes of GSH degradation, and GGT contributed more to GSH degradation than PepT. Furthermore, GGT activity was affected greatly by culture temperature, and the effect of GGT on GSH degradation could be eliminated by shortening the culture time at 30°C and extending the induction time at 42°C. However, the effect of PepT on GSH degradation could be eliminated only by disrupting the PepT gene. Finally, GSH degradation was not observed in GSH biosynthesis by E. coli JW1113 (pepT ⁻, pBV03), which was cultured at 30°C for 3 h and 42°C for 5 h. GSH concentration reached 15.60 mM, which was 2.19-fold of the control. To the best of our knowledge, this is the first report of prohibiting GSH degradation with PepT-deficient recombinant E. coli. The results are helpful to investigate the GSH metabolism pathway and construct a GSH biosynthesis system.     

Proteome expression patterns in the stress tolerant evergreen <Emphasis Type="Italic">Ammopiptanthus nanus</Emphasis> under conditions of extreme cold

Low temperature is one of the important environmental changes that affect plant growth. The cold resistance capabilities of evergreen plants are the result of long-term adaptation to extreme environmental conditions. To investigate the responses of Ammopiptanthus nanus, a rare stress-tolerant evergreen plant, to extreme cold stress, we analyzed the proteome expression patterns of stressed plants; this is the first study to report these patterns for A. nanus. We collected adult A. nanus leaves under two conditions of cold stress: extreme cold (−29°C) and relatively less extreme cold (−5°C). Total crude proteins were extracted from leaf blades, separated by two-dimensional gel electrophoresis, and stained with Coomassie brilliant blue. Of the 500 protein spots detected in each of the samples, eight of the spots that exhibited clear changes under the different conditions were identified by MALDI-TOF analyses. Our results suggest that cold stress-related proteins may play diverse roles in the resistance to multiple environmental stresses.     

Efficient condensed-phase production of perdeuterated soluble and membrane proteins

Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated media. Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression plasmids, can be condensed prior to protein expression, thereby providing high-quality ²H, ¹³C, ¹⁵N-enriched protein samples at 2.5–10% the cost of traditional methods. As an example of the value of such inexpensively-produced perdeuterated proteins, we produced ²H, ¹³C, ¹⁵N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40× condensed phase media, and obtained NMR spectra suitable for 3D structure determination. The cSPP system was also used to produce ²H, ¹³C, ¹⁵N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution. This allows a membrane protein’s structural and functional properties to be characterized prior to reconstitution, or as a probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a standardized protocol for production of perdeuterated proteins using the cSPP system. The 10–40 fold reduction in costs of fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated proteins in spectroscopic and crystallographic studies.     

Gene cloning,purification, and characterization of recombinant DNA ligases of the thermophilic archaea <Emphasis Type="Italic">Pyrococcus abyssi</Emphasis> and <Emphasis Type="Italic">Methanobacterium thermoautotrophicum</Emphasis>

DNA ligase genes of the thermophilic archaeae Pyrococcus abyssi (Pab DNA ligase) and Methanobacterium thermoautotrophicum (Mth DNA ligase) were cloned in Escherichia coli. The resulting recombinant enzymes were tested for activity in a ligation mixture with two oligonucleotides, one containing a preformed hairpin structure. The yield of the reaction products was maximal at temperatures close to 70°C for either enzyme; their accumulation reached a plateau at 70–75% of the theoretical yield at a stoichiometric enzyme-to-substrate ratio. The enzymes differed in thermal stability. The half-life of Pab DNA ligase was approximately 60 min at 90°C. Mth DNA ligase was completely inactivated within 10 min at this temperature. The recombinant DNA ligases from P. abyssi and M. thermoautotrophicum remained stabile during long-term storage at 4°C.     

Antibiotic translocation through membrane channels: temperature-dependent ion current fluctuation for catching the fast events

Temperature-dependent facilitated permeation of antibiotics through membrane channels was investigated. Here we reconstituted single OmpF trimers from the outer membrane of Escherichia coli (E. coli) into a planar lipid bilayer. The penetration of ampicillin through OmpF causes fluctuation in the ion current, and analysis of the fluctuations at different temperatures allows us to determine the mode of permeation. The residence time of the drug inside the channel decays strongly with temperature, reaching the resolution limit of the instrument at 30°C. The number of events increases exponentially with temperature up to 30°C and then gradually decreases as temperature increases. At room temperature, we observe about 25 events per second per monomer of the trimeric channel and an extrapolation to 37°C gives roughly 50 events. The activation energy for ampicillin translocation through OmpF is estimated to be around 13 kT. Temperature-dependent study gives new insights into the faster translocation of small substrates through biological nanopores.