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1.
Chinese Journal of Biotechnology ; (12): 4395-4405, 2021.
Article in Chinese | WPRIM | ID: wpr-921515

ABSTRACT

To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO), a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E. coli. Subsequently, the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification. The synthesized Bsppo gene contained 1 752 bp which encodes 585 amino acids with a deduced molecular weight of 65.3 kDa. Transformation of the recombinant vector into E. coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO. The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification. The results demonstrated that the optimal temperature and pH for BsPPO was 25°C and 4.0, respectively. BsPPO exhibited a good stability under low temperature and acidic environment. Low-intensity short-term light exposure increased the activity of BsPPO. Cu²⁺ could improve the activity of BsPPO while Zn²⁺ and Ca²⁺ showed the opposite effect. BsPPO could catalyze the oxidation of monophenols, diphenols, and triphenols, and exhibited good catalytic activity on l-tyrosine and vanillic acid. Moreover, BsPPO exhibited high catalytic activity on black sesame metabolites, including 2-methoxy cinnamic acid, indole-3-carboxylic acid and phloretin. These results may serve as a basis for further characterization of BsPPO.


Subject(s)
Catechol Oxidase/genetics , Cloning, Molecular , Escherichia coli/metabolism , Recombinant Proteins/genetics , Sesamum/genetics
2.
Chinese Journal of Biotechnology ; (12): 4124-4133, 2021.
Article in Chinese | WPRIM | ID: wpr-921493

ABSTRACT

The existence of cancer stem cells is regarded as the major cause for therapeutic resistance and relapse of a variety of cancer types including hepatocellular carcinoma (HCC). However, the tracing of such a subpopulation in vivo has been challenging. We have previously demonstrated that the isoform 5 of the voltage-gated calcium channel α2δ1 subunit, which can be recognized specifically by a monoclonal antibody 1B50-1, is a bona fide surface marker for HCC stem cells. Here we developed a strategy for optical imaging of α2δ1-positive cells by using a fusion protein containing the single chain variable fragment (scFv) of Mab1B50-1 and the luciferase NanoLuc which was tagged with Flag in the C-terminal. The scFv of Mab1B50-1 was fused to the N-terminal of NanoLucFlag using overlap PCR, and the recombinant fragment, which was named as 1B50-1scFv-NanoLucFlag, was subsequently cloned into a eukaryotic expression vector. The resulting construct was transfected into FreeStyle 293F cells in suspension using PEI reagent. The expression of the fusion protein was identified as a protein with molecular weight about 50 kDa by Western blotting. After purification by ANTI-FLAG® M2 affinity chromatography, 1B50-1scFv-NanoLucFlag was demonstrated to bind to α2δ1 positive cells specifically with a Kd value of (18.62±1.84) nmol/L. Furthermore, a strong luciferase activity of 1B50-1scFv-NanoLucFlag was detected in α2δ1 positive cells following incubation with the fusion protein, indicating that the presence of α2δ1 could be quantified using this fusion protein. Hence, 1B50-1scFv-NanoLucFlag provides a potential tool for optical imaging of α2δ1 positive cancer stem cells both in vitro and in vivo.


Subject(s)
Carcinoma, Hepatocellular , Humans , Liver Neoplasms , Neoplastic Stem Cells , Recombinant Proteins/genetics , Single-Chain Antibodies/genetics
3.
Chinese Journal of Biotechnology ; (12): 4083-4094, 2021.
Article in Chinese | WPRIM | ID: wpr-921489

ABSTRACT

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.


Subject(s)
Codon/genetics , Humans , Pichia/genetics , Recombinant Proteins/genetics , Saccharomycetales , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factors
4.
Chinese Journal of Biotechnology ; (12): 2786-2793, 2021.
Article in Chinese | WPRIM | ID: wpr-887841

ABSTRACT

To obtain chicken CD40L protein, the cDNA was prepared from chicken splenic cells and used as a template to clone and amplify CD40L by PCR. The target gene was cloned into pFastBac vector to construct a pFastBac-chCD40L donor plasmid. Recombinant plasmid was transformed into DH10Bac and recombinant Bacmid-chCD40L was obtained. The Bacmid-chCD40L plasmid was transfected into sf9 insect cells to obtain His-chCD40L protein. In addition, the target gene was cloned into pQM01 vector to construct a pQM01-chCD40L plasmid, recombinant plasmid was transfected into HEK 293T cells to obtain Strep-chCD40L protein. The chCD40L protein was purified by affinity chromatography, and the concentration of purified chCD40L protein was determined to be 0.01 mg/mL. Primary cells were isolated from the bursal tissue of 3-week old SPF chickens, and the chCD40L protein was added to the culture medium to stimulate cells. The chCD40L could bind to CD40 on B cells as examined by Western blotting, indirect immunofluorescence assay and flow cytometry, suggesting that chCD40L protein is biologically active. We successfully obtained chicken CD40L protein of biological activity, which laid the foundation in the in vitro culture of primary B lymphocytes for the isolation and diagnosis of virulent IBDV.


Subject(s)
Animals , Baculoviridae/genetics , CD40 Ligand/genetics , Chickens , Cloning, Molecular , Genetic Vectors/genetics , Recombinant Proteins/genetics
5.
Chinese Journal of Biotechnology ; (12): 1368-1375, 2021.
Article in Chinese | WPRIM | ID: wpr-878638

ABSTRACT

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.


Subject(s)
Bacterial Proteins , Diphtheria Toxin/genetics , Escherichia coli/genetics , Humans , Molecular Chaperones/genetics , Recombinant Proteins/genetics
6.
Chinese Journal of Biotechnology ; (12): 939-949, 2021.
Article in Chinese | WPRIM | ID: wpr-878605

ABSTRACT

Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.


Subject(s)
6-Phytase/genetics , Pichia/genetics , Plasmids , Recombinant Proteins/genetics , Saccharomycetales
7.
Chinese Journal of Biotechnology ; (12): 312-320, 2021.
Article in Chinese | WPRIM | ID: wpr-878564

ABSTRACT

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.


Subject(s)
Animals , CHO Cells , Cricetinae , Cricetulus , Enzymes/metabolism , Recombinant Proteins/genetics
8.
Chinese Journal of Biotechnology ; (12): 218-227, 2021.
Article in Chinese | WPRIM | ID: wpr-878556

ABSTRACT

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.


Subject(s)
Acetylglucosamine , Acetylglucosaminidase , Bacillus coagulans , Chitin , Chitinases , Hydrogen-Ion Concentration , Recombinant Proteins/genetics
9.
Chinese Journal of Biotechnology ; (12): 187-195, 2021.
Article in Chinese | WPRIM | ID: wpr-878553

ABSTRACT

In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.


Subject(s)
African Swine Fever/diagnosis , African Swine Fever Virus/genetics , Animals , Antibodies, Viral , Enzyme-Linked Immunosorbent Assay , Recombinant Proteins/genetics , Swine
10.
Braz. j. microbiol ; 49(4): 856-864, Oct.-Dec. 2018. graf
Article in English | LILACS | ID: biblio-974294

ABSTRACT

ABSTRACT The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 ºC + 1%M provided the most stable viability. By contrast, the temperature of 18 ºC + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.


Subject(s)
Pichia/metabolism , Fatty Acids, Nonesterified/metabolism , Pichia/growth & development , Pichia/genetics , Temperature , Recombinant Proteins/genetics , Culture Media/metabolism , Culture Media/chemistry , Methanol/metabolism , Fermentation , Glycerol/metabolism
11.
Braz. j. microbiol ; 49(3): 662-667, July-Sept. 2018. tab, graf
Article in English | LILACS | ID: biblio-951808

ABSTRACT

Abstract The effect of the intracellular microenvironment in the presence of an oxygen vector during expression of a fusion protein in Escherichia coli was studied. Three organic solutions at different concentration were chosen as oxygen vectors for fumarase expression. The addition of n-dodecane did not induce a significant change in the expression of fumarase, while the activity of fumarase increased significantly to 124% at 2.5% n-dodecane added after 9 h induction. The concentration of ATP increased sharply during the first 6 h of induction, to a value 7600% higher than that in the absence of an oxygen-vector. NAD/NADH and NADP/NADPH ratios were positively correlated with fumarase activity. n-Dodecane can be used to increase the concentration of ATP and change the energy metabolic pathway, providing sufficient energy for fumarase folding.


Subject(s)
Oxygen/metabolism , Gene Expression , Alkanes/metabolism , Escherichia coli/genetics , Fumarate Hydratase/metabolism , Oxygen/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Protein Folding , Alkanes/chemistry , Escherichia coli/metabolism , Fumarate Hydratase/genetics , Fumarate Hydratase/chemistry , NADP/metabolism , NADP/chemistry
12.
Braz. j. microbiol ; 49(3): 647-655, July-Sept. 2018. graf
Article in English | LILACS | ID: biblio-951810

ABSTRACT

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.


Subject(s)
Aspergillus fumigatus/enzymology , Fungal Proteins/genetics , Fungal Proteins/chemistry , Gene Expression , Cellulase/genetics , Cellulase/chemistry , Cloning, Molecular , Aspergillus fumigatus/genetics , Substrate Specificity , Enzyme Stability , Kluyveromyces/genetics , Kluyveromyces/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Fungal Proteins/metabolism , Cellulase/metabolism , Hot Temperature , Hydrogen-Ion Concentration
13.
Braz. j. microbiol ; 49(2): 414-421, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-889234

ABSTRACT

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.


Subject(s)
Antifungal Agents/pharmacology , Chitinases/pharmacology , Hordeum/enzymology , Recombinant Proteins/metabolism , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Blotting, Western , Chitinases/chemistry , Chitinases/genetics , Chitinases/isolation & purification , Chromatography, Affinity , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hordeum/genetics , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid
14.
Braz. j. microbiol ; 49(supl.1): 119-127, 2018. tab, graf
Article in English | LILACS | ID: biblio-974317

ABSTRACT

Abstract Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.


Subject(s)
Pichia/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Carbon/metabolism , Promoter Regions, Genetic , Pichia/growth & development , Pichia/metabolism , Temperature , Industrial Microbiology
15.
Braz. j. microbiol ; 49(supl.1): 68-75, 2018. tab, graf
Article in English | LILACS | ID: biblio-974318

ABSTRACT

Abstract Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp ® plate coated with 50 ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD450 nm) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle.


Subject(s)
Animals , Cattle , Enzyme-Linked Immunosorbent Assay/methods , Serologic Tests/methods , Enzootic Bovine Leukosis/diagnosis , Leukemia Virus, Bovine/immunology , Capsid Proteins/immunology , Antibodies, Viral/blood , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Enzyme-Linked Immunosorbent Assay/instrumentation , Sensitivity and Specificity , Enzootic Bovine Leukosis/blood , Enzootic Bovine Leukosis/virology , Leukemia Virus, Bovine/isolation & purification , Leukemia Virus, Bovine/genetics , Capsid Proteins/analysis , Capsid Proteins/genetics
16.
Braz. j. microbiol ; 48(4): 809-814, Oct.-Dec. 2017. graf
Article in English | LILACS | ID: biblio-889176

ABSTRACT

ABSTRACT This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.


Subject(s)
Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Bacillus subtilis/genetics , Genetic Vectors/genetics , Insect Proteins/genetics , Insect Proteins/metabolism , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Bacillus subtilis/metabolism , Escherichia coli/drug effects , Escherichia coli/growth & development , Gene Expression , Genetic Vectors/metabolism , Insect Proteins/isolation & purification , Insect Proteins/pharmacology , Protein Engineering , Protein Transport , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
17.
Braz. j. microbiol ; 48(3): 419-426, July-Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-889135

ABSTRACT

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.


Subject(s)
Pichia/metabolism , Industrial Microbiology/methods , Carbon/metabolism , Single-Chain Antibodies/biosynthesis , Antibodies/metabolism , Pichia/growth & development , Pichia/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Culture Media/metabolism , Culture Media/chemistry , Single-Chain Antibodies/genetics , Fermentation , Glycerol/metabolism , Lipoproteins, LDL/immunology , Antibodies/genetics
18.
Braz. j. microbiol ; 48(2): 286-293, April.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839389

ABSTRACT

Abstract This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.


Subject(s)
Humans , Pichia/metabolism , Recombinant Proteins/metabolism , Epidermal Growth Factor/metabolism , Pichia/genetics , Recombinant Proteins/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Cloning, Molecular , Culture Media/chemistry , Epidermal Growth Factor/genetics , Fermentation , Hydrogen-Ion Concentration
19.
Mem. Inst. Oswaldo Cruz ; 111(8): 535-538, Aug. 2016. graf
Article in English | LILACS | ID: lil-788999

ABSTRACT

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.


Subject(s)
Baculoviridae/chemistry , Baculoviridae/metabolism , Hepatitis A virus/chemistry , Viral Proteins/biosynthesis , Baculoviridae/genetics , Gene Expression Regulation, Viral , Genetic Vectors , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Solubility , Viral Proteins/chemistry , Viral Proteins/genetics
20.
Braz. j. microbiol ; 47(2): 394-402, Apr.-June 2016. tab, graf
Article in English | LILACS | ID: lil-780824

ABSTRACT

Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.


Subject(s)
Humans , Bacterial Proteins/analysis , Clostridioides difficile/genetics , Clostridium Infections/microbiology , Antibodies, Bacterial/analysis , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Gene Expression , Blotting, Western , Clostridioides difficile/isolation & purification , Clostridioides difficile/immunology , Clostridium Infections/diagnosis , Ribotyping , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology
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