Novel GFP expression using a short N-terminal polypeptide through the defined twin-arginine translocation (Tat) pathway

Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔGRNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.     

A novel expression system for recombinant marine mussel adhesive protein Mefp1 using a truncated OmpA signal peptide

To express an increased level of recombinant Mefp1 (marine mussel adhesive protein) in soluble form, we constructed expression vectors encoding truncated OmpA signal peptide-Mefp1 fusion proteins. OmpA signal peptide (OmpASP) is the 21 residue peptide fragment of the 23 residue OmpA signal sequence cleavable by signal peptidase I. We successfully produced increased levels of soluble recombinant Mefp1 (rMefp1) with various deletions of OmpASP, and found that the increased expression was caused by the increased pI of the N-terminus of the fusion proteins (> or = 10.55). All the OmpA signal peptide segments of 3-21 amino acids in length had the same pI value (10.55). Our results suggest that the pI value of the truncated OmpASP (OmpASP(tr)) play an important role in directional signaling for the fusion protein, but we found no evidence for the presence of a secretion enhancer in OmpASP. For practical applications, we increased the expression of soluble rMefp1 with OmpASP(tr) peptides as directional signals, and obtained rMefp1 with the native amino terminus (nN-rMefp1) using an OmpASP(tr)| Xa leader sequence that contains the recognition site for Xa protease.     

Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L⁻¹ production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.     

High level of expression of the Toxoplasma gondii-recombinant Rop2 protein in Escherichia coli as a soluble form for optimal use in diagnosis

The rhoptry 2 protein (Rop2) is an interesting protein of Toxoplasma gondii that is involved in the parasite invasion of host cell, it has three T-cell epitopes and high antigenic value. However, the expression of Rop2 as a recombinant protein in Escherichia coli is not an easy task, showing low levels of expression or degradation and solubility problems. Using a recombinant Rop2_196–561 fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth. rRop2_196–561 was purified mainly as a soluble product and in high concentrations (approx 1 mg/mL) under native conditions (40 mM imidazol in phosphate buffer). However, after a cycle of freezing-thawing rRop2_196–561 became insoluble. When glycerol was added to 26%, immediately after purification, the protein stayed soluble after cycles of freezing-thawing. Finally, it was demonstrated that under these conditions soluble rRop2_196–561 keeps its diagnostic value in contrast with the insoluble protein.     

Single pH buffer refolding screen for protein from inclusion bodies

We previously reported the set up of an automated test for screening the refolding of recombinant proteins expressed as inclusion bodies in Escherichia coli[1]. The screen used 96 refolding buffers and was validated with 24 proteins, 70% of which remained soluble in at least one buffer. In the present paper, we have analyzed in more detail these experimental data to see if the refolding process can be driven by general rules. Notably, we found that proteins with an acidic isoelectric point (pI) refolded in buffers the average pH of which was alkaline and conversely. In addition, the number of refolding buffers wherein a protein remained soluble increased with the difference between its pI and the average pH of the buffers in which it refolded. A trend analysis of the other variables (ionic strength, detergents, etc.) was also performed. On the basis of this analysis, we devised and validated a new refolding screen made of a single buffer for acidic proteins and a single buffer for alkaline proteins.     

Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.     

Molecular and biochemical characterization of a new alkaline β-propeller phytase from the insect symbiotic bacterium <Emphasis Type="Italic">Janthinobacterium</Emphasis> sp. TN115

A phytase-encoding gene (phyA115) was cloned from Janthinobacterium sp. TN115, a symbiotic bacterial strain isolated from the gut contents of Batocera horsfieldi larvae (Coleoptera: Cerambycidae), and expressed in Escherichia coli. The 1,884-bp full-length gene encodes a 28-residue putative signal peptide and a 599-residue mature protein with a calculated mass of 64 kDa. The deduced PhyA115 shares low identity with known sequences (47% at most) and contains an N-terminal incomplete domain (residues 29–297; domain N) and a typical β-propeller phytase domain at the C terminus (residues 298–627; domain C). Distinct from other β-propeller phytases that have neutral pH optima (pH 6.0–7.5), purified recombinant PhyA115 exhibits maximal activity at pH 8.5 and 45°C in the presence of 1 mM Ca²⁺ and is highly active over a wider pH range (pH 6.0–9.0). These results indicate that PhyA115 is a β-propeller phytase that has application potential in aquaculture feed. To our knowledge, this is the first report of cloning of a phytase gene from the symbiotic microbes of an insect digestive tract and from the genus Janthinobacterium. The N-terminal incomplete domain is found to have no phytase activity but can influence the pH property of PhyA115.     

Comparison of sialylation of maltase-glucoamylase in brush-border and soluble fractions of the small intestine of immature rats.

Two fractions of high-molar-mass soluble neutral maltase-glucoamylase (G1 and G2) of distal small intestine of 18-day-old rats separated on Sepharose 4B differ in sialylation which is reflected in their pI values obtained by chromatofocusing. The major soluble G1 fraction shows eight sialylated peaks converted by neuraminidase into a single fraction eluted at pH 4.21. Fraction G2 is less sialylated and neuraminidase causes its pI shift to 4.36. The chromatofocusing pattern suggests that G1 contains more acidic and G2 more basic glycoforms than their membrane-bound counterpart. Presence of less acidic pI values in the soluble G1 fraction of 18-day-old rats than in that of 13-day-old rats indicates that developmental decrease of sialylation concerns not only membrane-bound but also the soluble membrane-type of maltase-glucoamylase.     

High-level expression of active human alpha1-antitrypsin in transgenic tobacco chloroplasts

We have produced human alpha1-antitrypsin (A1AT), a major therapeutic protein, in genetically engineered tobacco plastids. Four different expression vectors have been evaluated which encode A1AT under the control of various 5′ and 3′ plastid expression elements. The use of heterologous promoter and terminator sequences derived from the corn and soybean plastid genomes leads to simpler and predictable recombinant genome patterns, avoiding unwanted recombination products between introduced and resident tobacco sequences. High level expression of unglycosylated A1AT, representing up to 2% of total soluble proteins, has been measured in leaves of transgenic tobacco lines. Some heterogeneity in the recombinant A1AT is detected after 2D protein separation, but the chloroplast-made protease inhibitors are fully active and bind to porcine pancreatic elastase.     

Habitat Use of Juvenile Fish in the Lower Danube and the Danube Delta: Implications for Ecotone Connectivity

Functional processes in freshwater ecosystems are highly influenced by acidic conditions. Foodwebs are affected and macroinvertebrate species diversity is decreased. This study aims to investigate leaf decomposition at very low pH in the acidic Banyupahit–Banyuputih river originating from the acidic crater lake Kawah Ijen in Indonesia. Leaf decomposition experiments were carried out for 200 days in the acidic river at pHs of approximately 0.7, 2.3 and 3.0 and in the neutral Kali Sengon river, using leaves from teak, Tectona grandis, and bamboo, Bambusa sp. Two different types of leaf packs were used: fine mesh size packs were used to exclude macroinvertebrates and coarse mesh size packs allowed macroinvertebrate colonization. Clear differences in decomposition rate were observed between the neutral Kali Sengon and the acidic Banyupahit–Banyuputih river with decomposition in the Kali Sengon river proceeding significantly faster for both leaf types. In the Kali Sengon k values (d⁻¹) over 46 days were 0.0202 for fine teak, 0.0236 for coarse teak, 0.0114 for fine bamboo and 0.0151 for coarse bamboo. No significant differences were observed between the three sites in the acidic Banyupahit–Banyuputih river with k values of 0.0034–0.0066 for fine teak, 0.0002–0.0057 for coarse teak, 0.0029–0.0054 for fine bamboo and 0.0000–0.0068 for coarse bamboo. Moreover, no clear adaptation of macroinvertebrates or microbes to low pH conditions could be detected. The coarse mesh leaf packs in the neutral Kali Sengon river revealed that macroinvertebrates are important in the breakdown process. Fine mesh packs revealed that microbial activity is depressed under acidic conditions. Based on this evidence, we conclude that the toxicity at low pH conditions, and probably also the precipitation of metals on the leaf material, seriously affects leaf decomposition.     

Artificial control of cytoplasmic pH and its bearing on cytoplasmic streaming,electrogenesis and excitability of characeae cells

Effects of changing the cytoplasmic pH on the cytoplasmic streaming, membrane potential and membrane excitability were studied in tonoplast-free cells ofChara australis andNitellopsis obtusa. The cytoplasmic pH was varied by internal perfusion of pH-buffered media.Nitellopsis cells were perfused only once, whileChara cells were perfused twice to control the pH more accurately. In both materials the rate of cytoplasmic streaming was maximum at about pH 7, low at pH 8.5–9 and almost zero at pH 5–5.5. The membrane potential was most negative at about pH 7. InChara the membrane potential supported by Mg·ATP was strongly inhibited at pH 5.5, and almost zero at pH 9, supporting the results obtained by Fujiiet al. (1979) on cells ofChara australis which were perfused once. The action potential could be induced by electrical stimulation inChara at pH 6.0–9.0 and inNitellopsis at pH 6.6–7.9. The membrane resistance ofNitellopsis was high at acidic and neutral pH values and low at alkaline pH, while that ofChara was low at both acidic and alkaline pH values.     

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.     

Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum

Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization–ion trap–mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60°C, thermal stability for 2 h at 40°C, optimum pH 3.5 (km=62 μM) measured on 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4–8 (75% stability at pH levels 2.2 and 9) for 2 h at 25°C.     

Simple and efficient expression of <Emphasis Type="Italic">Agaricus meleagris</Emphasis> pyranose dehydrogenase in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L⁻¹ PDH or 1,330 U L⁻¹ d⁻¹ in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown.     

Phylogenetic composition of bacterial communities in small boreal lakes and ombrotrophic bogs of the upper Volga basin

Fluorescent in situ hybridization (FISH) with rRNA-specific oligonucleotide probes was used to assess the numbers and phylogenetic diversity of prokaryotic microorganisms in the water of small boreal lakes and peatland catchments of the swampy upper Volga basin. The abundance of bacterioplankton in lake water was found to vary from 1.6 to 8.7 × 10⁶ cells ml⁻¹, with the highest values detected in neutral eutrophic lakes. The total cell numbers in the peat of ombrotrophic bogs were 3.9–4.3 × 10⁸ cells g⁻¹ of wet peat. The proportion of bacteria identified by the group-specific probes decreased from 79–85% in neutral (pH 6.6–6.9) mesotrophic and eutrophic lakes to 65–69% in acidic (pH 4.4–5.5) dystrophic lakes and to 51–58% in the peat of acidic (pH 3.6–3.9) ombrotrophic bogs. The diversity of bacterial communities was highest in lakes with neutral water. These communities were dominated by members of the phylum Actinobacteria (31–44% of the total bacterial number), while the contribution of Alphaproteobacteria (16–19%), Bacteroidetes (6–16%), Betaproteobacteria (6–7%), Planctomycetes (2–8%), and Gammaproteobacteria (4–5%) was also significant. In acidic dystrophic lakes, Actinobacteria (25–35%) and Betaproteobacteria (25–34%) predominated, while peatland catchments were dominated by the Alphaproteobacteria (20–23%). The presence of acidobacteria and some planctomycetes common for bogs in the water of acidic dystrophic lakes, as well as the high proportion of bacteria (31–49%) that were not identified by the group-specific probes, suggest the impact of microbial processes in peatland catchments on the microbial composition of the receiving waters.     

Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope

The DNA encoding soluble B lymphocyte stimulator (134–285 amino acids, sBLyS) mutant with residues 217–224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5′-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5α after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.     

Effect of Heat and pH Denaturation on the Structure and Conformation of Recombinant Human Hepatic Stimulator Substance

Hepatic stimulator substance (HSS) is a novel liver-specific growth-promoting factor. Although HSS has been successfully crystallized, several properties of this protein have yet to be determined. This study shows that recombinant human HSS (rhHSS) is a dimer with a molecular mass of 31 kDa, The protein is weakly acidic and has an isoelectric point (pI) of 4.50. rhHSS was able to protect hepatoma cells from H₂O₂-induced apoptosis and to stimulate cell growth. The recombinant protein was thermostable up to 80°C and resistant to changes in pH, as determined by synchronous fluorescence and far-UV circular dichroism (CD). Within the range of pH 4.0–10.0, rhHSS assumed a folded conformation identical to the secondary structure of the original, native protein and a native-like far-UV CD spectrum. Denatured rhHSS could be partly reconstituted with respect to its structure, but not its activity. Thus, rhHSS is a structurally stable protein insensitive to thermal and acid–alkaline denaturation.     

Biomass Enhancement in Maize and Soybean in Response to Glutamate Dehydrogenase Isomerization

The relationship between nutrient composition, crop biomass, and glutamate dehydrogenase (GDH) isoenzyme pattern was investigated in soybean (Glycine max) and maize (Zea mays) by monitoring the nutrient induced isomerization of the enzyme from the seedling stage to the mature crop. GDH was extracted from the leaves of the plants, and the isoenzymes were fractionated by isoelectric focusing followed by native polyacrylamide gel electrophoresis. The isomerization V_max values for soybean GDH, similar to maize GDH increased curvilinearly from 200 – 400 μmol mg⁻¹ min⁻¹ as the inorganic phosphate nutrient applied to the soil decreased from 50 − 0 mM. In soybean, combinations of N and K, P, or S nutrients induced the acidic and neutral isoenzymes, and gave biomass increases 25 – 50 % higher than the control plant. GDH isoenzymes were suppressed in soybean that received nutrients without N, K, or P and accordingly the biomass was about 30 % lower than the control. Treatment of maize with NPK nutrients increased the GDH V_max values from 138.9 at the vegetative to 256.4 μmol mg⁻¹ min⁻¹ at the reproductive phase, and suppressed the basic isoenzymes, but induced both the acidic and neutral isoenzymes thereby inducing seed production (27.0 ± 1.4 g per plant); whereas both the acidic and basic isoenzymes were suppressed in the control maize, and seeds did not develop. Simultaneous induction of the acidic, neutral, and basic isoenzymes of GDH indicated the occurrence of senescence. Therefore in maize and soybean, the induction of the acidic and basic isoenzymes of GDH led to the enhancement of biomass. This revised version was published online in July 2006 with corrections to the Cover Date.     

Proteins and Peptidases from Conidia and Mycelia of <Emphasis Type="Italic">Scedosporium apiospermum</Emphasis> Strain HLPB

The conidia–mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62–48 and 22–18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis. Martha Machado Pereira and Bianca Alcantara Silva contributed equally to this work.     

Purification and characterization of novel antifungal peptide,mouse beta defensin-1, in Escherichia coli

Mouse beta defensin-1 (mBD-1) is a cationic peptide with broad antimicrobial activity. The mBD-1 gene was cloned and fused with TrxA to construct pET32-mBD1, which was transformed into E. coli BL21 (DE3). The optimal expression conditions of fusion protein TrxA–mBD1 were: cultivation at 32°C in 2 × YT medium, induction with 0.2 mM isopropylthio-d-galactoside (IPTG), and post-induction expression for 8 h. The fusion protein was highly soluble (90.0%) and accounted for 65% of the total soluble protein; and its volumetric productivity reached 0.67 g/l, i.e., 0.14 g/l of recombinant mBD-1. At 5 μM, purified recombinant mBD-1 killed 50% of Candida albicans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.