One-step Affinity Chromatography Research Articles

Expression and characterization of β-1,3-1,4-glucanase of Aspergillus usamii in Escherichia coli and its application in sourdough bread making.

A β-1,3-1,4-glucanase gene (Auglu12A) from Aspergillus usamii was successfully expressed in Escherichia coli BL21(DE3). The recombinant enzyme, reAuglu12A was efficiently purified using the one-step nickel-nitrilotriacetic acid affinity chromatography. The specific activity of reAuglu12A was 694.8U/mg, with an optimal temperature of 55°C and pH of 5.0. The reAuglu12A exhibited stability at temperatures up to 60°C and within the pH range of 4.0-5.5. The reAuglu12A hydrolytic activity was increased in the presence of metal ions, especially K+ and Na+ , whereas it exhibited a Km and Vmax of 8.35mg/mL and 1254.02µmol/min/mg, respectively, toward barley β-glucan at pH 5.0 and 55°C. The addition of reAuglu12A significantly increased the specific volume (p<0.05) and reduced crumb firmness and chewiness (p<0.05) of wheat-barley sourdough bread during a 7-day storage period compared to the control. Overall, the quality of wheat-barley sourdough bread was improved after incorporation of reAuglu12A (especially at 3000U/300g). These changes were attributed to the synergistic effect of acidification by sourdough and its metabolites which provided a conducive environment for the optimal action of reAuglu12A in the degradation of β-glucans of barley flour in sourdough. This stabilized the dough structure, thereby enhancing the quality, texture, and shelf life of the bread. These findings suggest that reAuglu12A holds promise as a candidate for β-glucanase application in the baking industry.

Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11.

L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg-1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL-1 min-1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.

Expression and Purification of Recombinant T7 RNA Polymerase in Escherichia Coli

T7 RNA polymerase, DNA dependent RNA polymerase commonly employed in virus molecular research, in vitro transcription and virus replication. The aim of this study was to produce a recombinant T7 RNA polymerase enzyme employing an E. coli protein expression system. In addition, this project aimed to establish and optimize high-yield expression of soluble recombinant T7 RNA polymerase in E. coli cells. Escherichia coli are the most commonly used protein expression systems because of the ease of cultivation, high yield, and low cost. E. coli expression system based on T7 promoter (pRSET-a) was used for T7 RNA polymerase recombinant protein expression. The T7 RNAP gene was amplified and ligated into a pRSET-an expression vector with a hexa-histidine tag at the N-terminus. The ligated vector was effectively transformed into E. coli BL21 (DE3). For T7 RNAP recombinant protein overexpression, it was confirmed that T7 RNA polymerase gene could be induced with IPTG up to 1.0mM, when the OD600 of the culture broth reached 0.5-1.2. The recombinant T7 RNA polymerase was harvested from the whole cell lysate after three hours of induction at 18oC. T7 RNA polymerase recombinant protein was efficiently purified by one-step immobilized-metal affinity chromatography (IMAC) using Ni-NTA resins. SDS-PAGE and western blot analysis confirmed the identity of the purified T7 RNA polymerase recombinant protein in soluble fractions with purity in the range of 60%. T7 RNA polymerase recombinant protein expression can be achieved by considering specific factors and suitable conditions.

One-Shot Generation of Epitope-Directed Monoclonal Antibodies to Multiple Nonoverlapping Targets: Peptide Selection, Antigen Preparation, and Epitope Mapping.

This chapter describes an epitope-directed approach to generate antipeptide monoclonal antibodies to multiple nonoverlapping protein sites using a cocktail of fusion peptides as immunogen. It provides a step-by-step protocol on how antigenic peptides on a target protein can be identified by in silico prediction and discusses considerations for final peptide selection. Each antigenic peptide (10-20 amino acids long) is displayed as three-copy inserts on the surface exposed loop of a thioredoxin scaffold protein. The corresponding DNA coding sequence specifying the tripeptide insert flanked by Gly-Ser-Gly-Ser-Gly linkers is cloned in-frame into the Rsr II site of the thioredoxin gene in the pET-32a vector. The presence of a C-terminal polyhistidine tag (His6-tag) allows the soluble fusion proteins to be purified by one-step native immobilized metal affinity chromatography (IMAC) to greater than 95% purity. Multiple thioredoxin fusion proteins are mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The use of short antigenic peptides of known sequence facilitates direct epitope mapping requiring only small mutagenesis scan peptide libraries in the multipin peptide format.

Applications of Cell-Free Synthesized Membrane Protein Precipitates.

Cell-free protein expression systems are new core platforms for membrane protein synthesis. Expression in thepresence of supplied artificial hydrophobic environments such as nanomembranes or micelles allows the co-translational solubilization and folding of membrane proteins. In theabsence of hydrophobic compounds, the synthesized membrane proteins quantitatively precipitate, while frequently still retaining a significant part of folded structural elements. This so-called precipitate-forming cell-free (P-CF) expression mode is avery effective and reliable approach for numerous applications. Even from complex membrane proteins such as G-protein coupled receptors or large transporters, significant amounts of such precipitates can be synthesized within few hours. The precipitates can be solubilized in detergents or reconstituted into membranes for subsequent structural or functional analysis. Harsh denaturation and refolding procedures as known from the treatment of bacterial inclusion bodies are usually not required.This strategy is particularly interesting for applications requiring large amounts of membrane protein or fast access to a sample. It is further an excellent tool for the production of membrane protein antigens suitable for antibody generation. The purification of the precipitates in downstream processing is streamlined as only few proteins from the cell-free lysate may co-precipitate with the synthesized membrane protein. For most applications, a one-step affinity chromatography by taking advantage of small purification tags attached to the membrane protein target is sufficient. We give an overview on current applications of P-CF precipitates and describe the underlying techniquesin detail. We furthermore provide protocols for the successful crystallization and NMR analysis of P-CF synthesized membrane proteins exemplified with the diacylglycerol kinase (DAGK). In addition, we describe the functional characterization of a P-CF synthesized large eukaryotic transporter.

Virus-like particles with FLAG-tagged envelope protein as a tetravalent dengue vaccine candidate

The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.

Fabrication and characterization of purified esterase-embedded zeolitic imidazolate frameworks for the removal and remediation of herbicide pollution from soil

The fragility and high cost of enzymes represent critical challenges limiting their practical application in the removal of pesticides. Herein, an aryloxyphenoxypropionate herbicide-hydrolyzing enzyme, QpeH, was purified via one-step affinity chromatography and embedded into two types of zeolitic imidazolate frameworks (ZIFs) through biomimetic mineralization. The catalytic activity towards the herbicide quizalofop-P-ethyl, the loading capacity and efficiency of the resulting two composites, QpeH@ZIF-10 with cruciate flower-like morphology and QpeH@ZIF-8 with rhombic dodecahedral morphology, were compared. Both composites had excellent stability and reusability after 10 reuse cycles, with QpeH@ZIF-10 having a better performance. More importantly, when applied for the removal of quizalofop-P-ethyl pollution in the watermelon field, QpeH@ZIF-10 (88%) showed a remarkably improved degradation efficiency compared to QpeH@ZIF-8 (84%) despite the latter having a greater loading capacity. Finally, the use of QpeH@ZIF composites was shown to recover the bacterial community in soil. This work provides a new insight into the low-cost synthesis of nanobiocatalysts combining simple purified enzymes and metal-organic frameworks (MOFs) for the remediation of pesticide-contaminated soils.

Phytocatalytic and cytotoxic activity of the purified laccase from bled resin of Pistacia atlantica Desf.

This study reports an efficient and fast procedure for the purification of laccase (PaL) obtained from the resin of Pistacia atlantica Desf. It was purified by one-step affinity chromatography and showed the specific activity of 393 U/mg with 81.9-fold purification. The molecular weight of PaL was estimated to be approximately 60 kDa using gel electrophoresis SDS-PAGE. Moreover, it depicted diphenolase activity and high affinity towards 2,6-dimethoxy phenol (Km = 10.01 ± 0.5 mM) and syringaldazine (Km = 6.57 ± 0.2 mM) comparing with plant-origin polyphenol oxidases reported in the literature. It should be noted that PaL possessed optimal activity at pH 7.5 and 45 °C. It also remained stable under different conditions of pH (6.5–8.0), temperature (25–45 °C), and when it was exposed to several metal ions. The MTT and flow cytometry assays demonstrated that the enzyme treatment significantly affected growth of HeLa, HepG2, and MDA-MB-231 cells with LC50 values of 4.83 ± 0.02, 61 ± 0.31, and 26.83 ± 0.11 μM after 72 h, respectively. Novelty statementThis is the first attempt to isolate and characterize a new oxidoreductase from the resin of Pistacia atlantica Desf., native species of Iran, to recruit it in cytotoxicity researches. In the purification process by an efficient affinity column (SBA-NH2-GA), the enzyme was eluted promptly with a satisfied yield. The purified laccase exerted higher affinity to diphenolic compounds and pH-thermal stability compared to other plant-derived polyphenol oxidases. The purified enzyme was found to show anti-oxidant capacity and significantly inhibited the growth of cancerous cells in vitro. PaL showed more cytotoxic activity towards HeLa and MDA-MB-231 cells by induction of apoptosis. The cytotoxic activity of the laccase was measured by flow cytometry.

Cloning, expression and biochemical characterization of a novel amidase from Thauera sinica K11

A novel amidase (TAM) was identified and cloned from the genome of Thauera sinica K11. The recombinant protein was purified to homogeneity by one-step affinity chromatography for up to 26.4-fold with a yield of 38.1%. Gel filtration chromatography and SDS-PAGE revealed that the enzyme was a tetramer with a subunit of approximately 37.5 kDa. The amidase exhibited the maximum acyl transfer activity at 45 °C and pH 7.0, and it was highly stable over a wide pH range of 6.0–11.0. Inhibition of enzyme activity was observed in the presence of metal ions, thiol reagents and organic solvents. TAM showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides. For linear aliphatic monoamides, the acyl transfer activity of TAM was decreased with the extension of the carbon chain length, and thus the highest activity of 228.2 U/mg was obtained when formamide was used as substrate. This distinct selectivity of amidase to linear aliphatic monoamides expanded the findings of signature amidases to substrate specificity.

Mass production of active recombinant Chryseobacterium proteolyticum protein glutaminase in Escherichia coli using a sequential dual expression system and one‐step purification

Protein glutaminase (PG) is an enzyme that specifically catalyzes the deamidation of glutamine residues on proteins or peptides, remarkably improving the solubility, emulsification and foaming properties of food proteins and, thereby, conferring great potential in food industry applications. PG is primarily produced from wild strains of Chryseobacterium proteolyticum and the low enzyme production yield restricts large-scale industrial applications. In this context, by evaluating different cleavage site insertions between the pro-region and mature domain of PG as well as different linkers flanking the cleavage site, an E. coli expression and purification protocol has been developed to produce active recombinant PG. To simplify the production workflow, we developed a sequential dual expression system. More than 15 mg of pure and active PG was obtained from 1 L of shaking-flask bacteria culture by one-step nickel affinity chromatography purification. The enzymatic characteristics of the recombinant PG protein were similar to those of native PG. For the deamidation effect of recombinant PG, the deamidation degree (DD) of gliadin reached up to 67% and the solubility increased 84-fold. Thus, this study provides a practical approach to mass producing active PG proteins and investigates its potential applications on food proteins.

Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carrying multiple copies of target gene

Introduction: Fibroblast growth factor-2 (FGF-2) is a multifunctional protein that plays an important role in the regulation of proliferation, differentiation and migration of a variety of cells. The recombinant human FGF-2 (rhFGF-2) is currently used in stem cell culture, medicine and cosmetic products. In this study, we aim to produce secreted rhFGF-2 protein from a Pichia pastoris strain containing multiple copies of the fgf-2 gene to eliminate the disadvantages of intracellular expression systems.&#x0D; Methods: The recombinant Pichia pastoris carrying the fgf-2 gene was cloned by using homologous cloning method. The recombinant strains were screened by PCR reactions using specific primers for the target gene and the AOX1 promoter sequence. Moreover, the copy number of the fgf-2 gene inserted into the P. pastoris genome was identified by semi-quantitative PCR method. The secreted rhFGF-2 protein was collected in the induced BMMY medium at a final methanol concentration of 0.5%, and purified by one-step heparin affinity chromatography. The quantity and biological activity of the purified protein were determined by competitive ELISA method and MTT assay on NIH-3T3 cell line, respectively.&#x0D; Results: Various recombinant P. pastoris clones carrying different copy numbers of the fgf-2 gene were obtained and categorized into 3 groups: the low copy strains (4-5 copies), medium copy strains (8-11 copies), and high copy strains (more than 20 copies). Among those strains, the 4-copy one produced the rhFGF-2 protein at the highest expression level. After purification, the purity of rhFGF-2 protein reached 98.8%, and the recovery yield was 179.2 µg of protein from 200 mL of flask culture (equivalent to 850 µg/L). The purified rhFGF-2 protein showed similar biological activity on NIH-3T3 cell line with the commercial FGF-2 protein.&#x0D; Conclusion: The recombinant FGF-2 protein was successfully secretory expressed from Pichia pastoris, and successfully purified by only one-step chromatography.

Functional Expression and One-Step Protein Purification of Manganese Peroxidase 1 (rMnP1) from Phanerochaete chrysosporium Using the E. coli-Expression System.

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn2+ to Mn3+, a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli-expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn2+.

Expression, Characterisation and Homology Modelling of a Novel Hormone-Sensitive Lipase (HSL)-Like Esterase from Glaciozyma antarctica

Microorganisms, especially those that survive in extremely cold places such as Antarctica, have gained research attention since they produce a unique feature of the protein, such as being able to withstand at extreme temperature, salinity, and pressure, that make them desired for biotechnological application. Here, we report the first hormone-sensitive lipase (HSL)-like esterase from a Glaciozyma species, a psychrophilic yeast designated as GlaEst12-like esterase. In this study, the putative lipolytic enzyme was cloned, expressed in E. coli, purified, and characterised for its biochemical properties. Protein sequences analysis showed that GlaEst12 shared about 30% sequence identity with chain A of the bacterial hormone-sensitive lipase of E40. It belongs to the H group since it has the conserved motifs of Histidine-Glycine-Glycine-Glycine (HGGG)and Glycine-Aspartate-Serine-Alanine-Glycine (GDSAG) at the amino acid sequences. The recombinant GlaEst12 was successfully purified via one-step Ni-Sepharose affinity chromatography. Interestingly, GlaEst12 showed unusual properties with other enzymes from psychrophilic origin since it showed an optimal temperature ranged between 50–60 °C and was stable at alkaline pH conditions. Unlike other HSL-like esterase, this esterase showed higher activity towards medium-chain ester substrates rather than shorter chain ester. The 3D structure of GlaEst12, predicted by homology modelling using Robetta software, showed a secondary structure composed of mainly α/β hydrolase fold, with the catalytic residues being found at Ser232, Glu341, and His371.

Heterologous expression and characterization of a novel serine protease from Daphnia magna: A possible role in susceptibility to toxic cyanobacteria

Mass developments of toxin-producing cyanobacteria are frequently observed in freshwater ecosystems due to eutrophication and global warming. These mass developments can partly be attributed to cyanobacterial toxins, such as protease inhibitors (PIs), which inhibit digestive serine proteases of Daphnia, the major herbivore of phytoplankton and cyanobacteria. To date, mechanisms of this inhibition in the gut of the crustacean Daphnia magna are not known. Here, we characterize a single serine protease, chymotrypsin 448 (CT448), which is present in the gut of the crustacean D. magna. Sequence alignments with human serine proteases revealed that CT448 has a putative N-terminal pro-peptide which is extended compared to the mammalian homologs and within this pro-peptide two N-linked glycosylation motifs were found. CT448 was heterologously expressed in Sf21 insect cells using a baculovirus expression system for optimized protein production and secretion into the medium. The protein was purified via a one-step affinity chromatography, which resulted in a protein yield of 3.45 mg/l medium. The inactive precursor (zymogen) could be activated by tryptic digestion. This is the first example of a recombinant expression of an active crustacean serine protease, which functions in the gut of Daphnia. Proteomic identification of protease cleavage sites (PICS) and hydrolysation of various synthetic substrates showed that CT448 is a chymotrypsin-like elastase. In this study, we confirm that CT448 is a target of cyanobacterial protease inhibitors. Local evolutionary modifications of CT448 might render this proteolytic enzyme less susceptible against cyanobacterial secondary metabolites and might improve the fitness of Daphnia during cyanobacterial blooms.

Bioconversion of inulin to difructose anhydride III by a novel inulin fructotransferase from Arthrobacter chlorophenolicus A6

The gene encoding difructose anhydride III (DFA III)-forming inulin fructotransferase (IFTase) from Arthrobacter chlorophenolicus A6 was cloned and overexpressed in Escherichia.coli. The recombinant IFTase (DFA III-forming) was purified using one-step nickel affinity chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses, the enzyme showed a homotrimeric form composed of three identical subunits, each with an apparent molecular mass of 43 kDa. The maximum catalytic activity was shown at 65 °C and pH 5.5, and the specific activity was measured as 902 U mg−1. The enzyme showed remarkable thermostability and over half of the initial activity was retained after incubation at 80 °C for 1 h. The Km and Vmax were estimated to 12.93 mM and 2.89 μmol min−1 ml−1, respectively. After complete hydrolysis of inulin for DFA III production by the purified enzyme, the produced minor products contained sucrose (GF), 1-kestose (GF2), and nystose (GF3). The smallest substrate was identified to be GF3. When 50, 100, and 200 g l−1 of inulin were hydrolyzed by the purified IFTase, the DFA III yield reached 81%, 72%, and 67%, respectively.

Generation of a recombinant Aggregatibacter actinomycetemcomitans RTX toxin in Escherichia coli

A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double–His6) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli. The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart.

Expression of Mastoparan B, a Venom Peptide, Via Escherichia coli C43 (DE3) Coupled with an Artificial Oil Body-Cyanogen Bromide Technology Platform.

Mastoparan B (MPB) is a venom peptide isolated from Vespa basalis (black-bellied hornet), one of the dangerous vespine wasps found in Taiwan. MPB is a tetradecapeptide (LKLKSIVSWAKKVL), amphiphilic venom peptide, with a molecular mass of 1.6 kDa. MPB belongs to an evolutionarily conserved component of the innate immune response against microbes. In this study, we attempted to modify a reliable oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB. The aim of this study was to develop an artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce the bioactive form of mastoparan B (MPB), which in a manner identical to that of its native counterpart. The plasmid pET30-His6-rOle(127M→L)-MPB was constructed, and then four different E. coli strains- BL21(DE3), BL21(DE3)pLysS, C41(DE3), and C43(DE3) were tested to identify the most suitable host for the pET30-His6-rOle(127M→L)-MPB fusion protein expression. We optimized the expression conditions by testing different growth temperatures, isopropyl-β-D-thiogalactoside (IPTG) concentrations, and post-induction collection times. Afterwards, the His6-rOle(127M→L)-MPB protein was purified by one-step nickel-chelated affinity chromatography (Ni2+-NTA) under denaturing conditions. The purified His6-rOle(127M→L)-MPB was selectively cleaved by thrombin protease to remove the His6-tag and the leader peptide from the N-terminus. Subsequently, rOle(127M→L)-MPB protein was constituted into AOB and incubated with CNBr for a cleavage reaction, which resulted in the release of the MPB from rOle(127M→L)-MPB protein via AOB. The purified MPB was identified by MALDI-MS and HPLC analysis, and its bioactivity was examined by antimicrobial testing. After a 2-h induction period, the E. coli C43(DE3) was found to be superior to BL21(DE3) and the other protease-deficient strains as an expression host. And, the optimal His6-rOle(127M→L)-MPB expression at 37°C for 2 h after induction with 5 µM IPTG. The purified MPB showed that a single major peak was detected by HPLC/UV detection with a retention time of 22.5 minutes, which was approximately 90% pure. The putative MPB, and over two-third of the peptide sequence was verified by the MALDI-MS analysis. Finally, the purified MPB was examined by a broth dilution-antimicrobial susceptibility test. These results indicated that the purified MPB was bioactive and very effective in anti-bacterial (E. coli J96) activity. Here, we successfully used the oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB peptide which, in a manner identical to that of its native counterpart. In this study, the recombinant oleosin based fusion strategy coupled with AOB-CNBr purification platform open a new avenue for the production of active MPB and facilitate the studies and applications of the peptide in the future for medicinal applications such as hypotension and antibacterial effect.

Expression, purification and function of cysteine desulfurase from Sulfobacillus acidophilus TPY isolated from deep-sea hydrothermal vent.

The cysteine desulfurase (SufS) gene of Sulfobacillus acidophilus TPY, a Gram-positive bacterium isolated from deep-sea hydrothermal vent, was cloned and over-expressed in E. coli BL21. The recombinant SufS protein was purified by one-step affinity chromatography. The TPY SufS contained a well conserved motif RXGHHCA as found in that of other microorganisms, suggesting that it belonged to group II of cysteine desulfurase family. The recombinant TPY SufS could catalyze the conversion of l-cysteine to l-alanine and produce persulfide, and the enzyme activity was 95μ/μL of sulfur ion per minute. The growth of E. coli BL21 was promoted by over-expressing TPY SufS in vivo or by directly adding recombinantTPY SufS in the medium (4.3-4.5×108cells/mL vs. 3.2-3.5×108cells/mL). Furthermore, the highest cell density of E. coli BL21 when the TPY SufS was over-expressed was about 3.5 times that of the control groups in the presence of sodium thiosulfate. These results indicate that the SUF system as the only assembly system of iron-sulfur clusters not only has significant roles in survival of S. acidophilus TPY, but also might be important for combating with high content of sulfide.

SHuffle™ T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties

BackgroundHuman fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1. MethodsA synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts (BL21 (DE3), Rosetta-gami™ 2(DE3), SHuffle™ T7). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties. ResultsThe highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84ng/mL. ConclusionAlthough SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.

Heterologous expression of a Streptomyces cyaneus laccase for biomass modification applications

Laccases are used for the conversion of biomass into fermentable sugars but it is difficult to produce high yields of active laccases in heterologous expression systems. We overcame this challenge by expressing Streptomyces cyaneus CECT 3335 laccase in Escherichia coli (ScLac) and we achieved a yield of up to 104 mg L−1 following purification by one-step affinity chromatography. Stability and activity assays using simple lignin model substrates showed that the purified enzyme preparation was active over a broad pH range and at high temperatures, suggesting it would be suitable for biomass degradation. The reusability of ScLac was also demonstrated by immobilizing the enzyme on agarose beads with a binding yield of 33%, and by the synthesis of cross-linked enzyme aggregates with an initial activity recovery of 72%.