Pub Date : 2016-03-01DOI: 10.1080/10826068.2016.1155060
L. F. Garcia, Stephen Rathinaraj Benjamin, R. Antunes, F. M. Lopes, V. Somerset, E. Gil
ABSTRACT A new strategy for the construction of a polyphenol oxidase carbon paste biosensor for paracetamol detection is reported. The eggplant (Solanum melongena) was processed to collect the polyphenol oxidase as an enzyme that was incorporated in the carbon paste sensor construction. The constructed sensor displayed high sensitivity and good selection for paracetamol detection and recognition. Optimized conditions included pH 6.0 (highest activity), pH 7.0 (highest stability), pulse amplitude of 50 mV, and 15% of vegetable extract per carbon paste. The sensor displayed a linear range from 20 to 200 µM, with a detection limit of 5 µM. Application of the sensor to paracetamol determination in tablet and oral solutions have shown satisfactory results. The efficiency of the method showed very good repeatability ranging between 1.26 and 1.72% relative standard deviation for interday analysis, while recoveries for paracetamol varied between 97.5 and 99.8% for the voltammetric determination. The strategy for a simple, low cost, and efficient eggplant polyphenol oxidase sensor showcased in this work provides an opportunity for the detection of other phenolic compounds in various matrices.
{"title":"Solanum melongena polyphenol oxidase biosensor for the electrochemical analysis of paracetamol","authors":"L. F. Garcia, Stephen Rathinaraj Benjamin, R. Antunes, F. M. Lopes, V. Somerset, E. Gil","doi":"10.1080/10826068.2016.1155060","DOIUrl":"https://doi.org/10.1080/10826068.2016.1155060","url":null,"abstract":"ABSTRACT A new strategy for the construction of a polyphenol oxidase carbon paste biosensor for paracetamol detection is reported. The eggplant (Solanum melongena) was processed to collect the polyphenol oxidase as an enzyme that was incorporated in the carbon paste sensor construction. The constructed sensor displayed high sensitivity and good selection for paracetamol detection and recognition. Optimized conditions included pH 6.0 (highest activity), pH 7.0 (highest stability), pulse amplitude of 50 mV, and 15% of vegetable extract per carbon paste. The sensor displayed a linear range from 20 to 200 µM, with a detection limit of 5 µM. Application of the sensor to paracetamol determination in tablet and oral solutions have shown satisfactory results. The efficiency of the method showed very good repeatability ranging between 1.26 and 1.72% relative standard deviation for interday analysis, while recoveries for paracetamol varied between 97.5 and 99.8% for the voltammetric determination. The strategy for a simple, low cost, and efficient eggplant polyphenol oxidase sensor showcased in this work provides an opportunity for the detection of other phenolic compounds in various matrices.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"4 1","pages":"850 - 855"},"PeriodicalIF":0.0,"publicationDate":"2016-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78482262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-09DOI: 10.1080/10826068.2016.1141299
S. Shukla, A. Verma, I. Kajala, Antti Nyyssolä, Rwivoo Baruah, K. Katina, R. Juvonen, M. Tenkanen, A. Goyal
ABSTRACT The dextransucrase gene from Weissella confusa Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in Lactococcus lactis. The recombinant dextransucrase, WcCab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. WcCab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. WcCab3-rDSR gave Km of 6.2 mM and Vm of 6.3 µmol/min/mg. The characterization of dextran synthesized by WcCab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of WcCab3-rDSR using the crystal structures of dextransucrase from Lactobacillus reuteri (protein data bank, PDB id: 3HZ3) and Streptococcus mutans (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (β/α)8 triose phosphate isomerase-barrel fold making the catalytic core of WcCab3-rDSR. The structure superposition and multiple sequence alignment analyses of WcCab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.
{"title":"Structure modeling and functional analysis of recombinant dextransucrase from Weissella confusa Cab3 expressed in Lactococcus lactis","authors":"S. Shukla, A. Verma, I. Kajala, Antti Nyyssolä, Rwivoo Baruah, K. Katina, R. Juvonen, M. Tenkanen, A. Goyal","doi":"10.1080/10826068.2016.1141299","DOIUrl":"https://doi.org/10.1080/10826068.2016.1141299","url":null,"abstract":"ABSTRACT The dextransucrase gene from Weissella confusa Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in Lactococcus lactis. The recombinant dextransucrase, WcCab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. WcCab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. WcCab3-rDSR gave Km of 6.2 mM and Vm of 6.3 µmol/min/mg. The characterization of dextran synthesized by WcCab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of WcCab3-rDSR using the crystal structures of dextransucrase from Lactobacillus reuteri (protein data bank, PDB id: 3HZ3) and Streptococcus mutans (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (β/α)8 triose phosphate isomerase-barrel fold making the catalytic core of WcCab3-rDSR. The structure superposition and multiple sequence alignment analyses of WcCab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"53 1","pages":"822 - 832"},"PeriodicalIF":0.0,"publicationDate":"2016-02-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83580669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
ABSTRACT The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.
{"title":"Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia","authors":"Chunsheng Hu, Xiaochen Cheng, Yuxin Lu, Zu-ze Wu, Qingling Zhang","doi":"10.1080/10826068.2016.1141302","DOIUrl":"https://doi.org/10.1080/10826068.2016.1141302","url":null,"abstract":"ABSTRACT The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"11 1","pages":"844 - 849"},"PeriodicalIF":0.0,"publicationDate":"2016-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74001969","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-06DOI: 10.1080/10826068.2016.1141301
V. Sant'anna, Ana Paula Folmer Correa, Amanda de Souza da Motta, A. Brandelli
ABSTRACT Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH4)2SO4], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH4)2SO4 system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH4)2SO4 and 1.0 mol l−1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.
{"title":"Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems","authors":"V. Sant'anna, Ana Paula Folmer Correa, Amanda de Souza da Motta, A. Brandelli","doi":"10.1080/10826068.2016.1141301","DOIUrl":"https://doi.org/10.1080/10826068.2016.1141301","url":null,"abstract":"ABSTRACT Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH4)2SO4], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH4)2SO4 system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH4)2SO4 and 1.0 mol l−1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"128 1","pages":"838 - 843"},"PeriodicalIF":0.0,"publicationDate":"2016-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87946001","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-06DOI: 10.1080/10826068.2016.1141300
Wenyan Zou, Xiaoyu Liu, Dian-hua Chen, Jie Wang, Xi Zhao, Jiahuang Li, Lina Ji, Z. Hua
ABSTRACT Based on their nanocage architectures, ferritins show their potential applications in medical imaging and therapeutic delivery systems. However, the recombinant human H-chain ferritin (rHF) is prone to form inclusion bodies in Escherichia coli. In our study, the cDNA of rHF was cloned into plasmid pET28a under the control of a T7 promoter. Molecular chaperones, including GroES, GroEL, and trigger factor, were coexpressed with rHF to facilitate its correct folding. The results showed that the solubility of rHF was increased more than threefold with the help of molecular chaperones. Taking advantages of its N-terminal His-tag, rHF was then purified with Ni-affinity chromatography. With a yield of 15 mg/L from bacterial culture, the purified rHF was analyzed by circular dichroism spectrometry for its secondary structure. Moreover, the rHF nanocages were characterized by transmission electron microscopy and dynamic light scattering. Our results indicate that rHF is able to self-assemble into nanocages with a narrow size distribution.
{"title":"Expression, purification, and characterization of recombinant human H-chain ferritin","authors":"Wenyan Zou, Xiaoyu Liu, Dian-hua Chen, Jie Wang, Xi Zhao, Jiahuang Li, Lina Ji, Z. Hua","doi":"10.1080/10826068.2016.1141300","DOIUrl":"https://doi.org/10.1080/10826068.2016.1141300","url":null,"abstract":"ABSTRACT Based on their nanocage architectures, ferritins show their potential applications in medical imaging and therapeutic delivery systems. However, the recombinant human H-chain ferritin (rHF) is prone to form inclusion bodies in Escherichia coli. In our study, the cDNA of rHF was cloned into plasmid pET28a under the control of a T7 promoter. Molecular chaperones, including GroES, GroEL, and trigger factor, were coexpressed with rHF to facilitate its correct folding. The results showed that the solubility of rHF was increased more than threefold with the help of molecular chaperones. Taking advantages of its N-terminal His-tag, rHF was then purified with Ni-affinity chromatography. With a yield of 15 mg/L from bacterial culture, the purified rHF was analyzed by circular dichroism spectrometry for its secondary structure. Moreover, the rHF nanocages were characterized by transmission electron microscopy and dynamic light scattering. Our results indicate that rHF is able to self-assemble into nanocages with a narrow size distribution.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"15 1","pages":"833 - 837"},"PeriodicalIF":0.0,"publicationDate":"2016-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74596787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-02DOI: 10.1080/10826068.2015.1045610
Hongyan Mao, Xiaofei Zhao, Hongjuan Zhu, Jingxia Guo, Zhenghai Ma
ABSTRACT Herpes simplex virus type 1 (HSV-1) is responsible for cold sores in the general population, but also contributes to the development of other more serious diseases in some circumstances. The viral glycoprotein D (gD) is essential for virus entry into host cells. In the present study, the Drosophila melanogaster Schneider 2 (S2) expression system (DES) was evaluated for the expression of recombinant gD1. The DNA sequences encoding the full-length gD1 (369aa, FLgD1) and a truncated gD1 form corresponding to the ectodomain (314aa, EgD1) were cloned into S2 expression vector pMT/BiP/V5-HisA to generate pMT-EgD1 and pMT-FLgD1, respectively. Two forms of gD1 gene were fitted with a hexahistidine tag to facilitate their purification. Cell populations expressing the highest gD1 levels were selected by using a limiting dilution assay. Western blot, flow cytometry (FACS), and confocal immunofluoresence assay demonstrated that the full-length form is restrained in the lipid membranes of the cell and the ectodomain form is secreted into the medium. Recombinant ectodomain gD1 was scaled up and purified from the culture medium using nickel nitrilotriacetic acid affinity chromatography, and a maximum production level of 56.8 mg/L of recombinant gD1 was obtained in a shake-flask culture of S2 cells after induction with 5 µM CdCl2 for 4 days. Mice were then immunized with recombinant purified gD1 and produced high titers of antibody measured by enzyme-linked immunosorbent assay (ELISA; 1:5,120,000) as well as high plaque neutralization titer (1:320). Overall, the data indicated that stable expression in S2 cells is a practical way of synthesizing gD1 for use in structural and functional studies in the further study.
{"title":"Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells","authors":"Hongyan Mao, Xiaofei Zhao, Hongjuan Zhu, Jingxia Guo, Zhenghai Ma","doi":"10.1080/10826068.2015.1045610","DOIUrl":"https://doi.org/10.1080/10826068.2015.1045610","url":null,"abstract":"ABSTRACT Herpes simplex virus type 1 (HSV-1) is responsible for cold sores in the general population, but also contributes to the development of other more serious diseases in some circumstances. The viral glycoprotein D (gD) is essential for virus entry into host cells. In the present study, the Drosophila melanogaster Schneider 2 (S2) expression system (DES) was evaluated for the expression of recombinant gD1. The DNA sequences encoding the full-length gD1 (369aa, FLgD1) and a truncated gD1 form corresponding to the ectodomain (314aa, EgD1) were cloned into S2 expression vector pMT/BiP/V5-HisA to generate pMT-EgD1 and pMT-FLgD1, respectively. Two forms of gD1 gene were fitted with a hexahistidine tag to facilitate their purification. Cell populations expressing the highest gD1 levels were selected by using a limiting dilution assay. Western blot, flow cytometry (FACS), and confocal immunofluoresence assay demonstrated that the full-length form is restrained in the lipid membranes of the cell and the ectodomain form is secreted into the medium. Recombinant ectodomain gD1 was scaled up and purified from the culture medium using nickel nitrilotriacetic acid affinity chromatography, and a maximum production level of 56.8 mg/L of recombinant gD1 was obtained in a shake-flask culture of S2 cells after induction with 5 µM CdCl2 for 4 days. Mice were then immunized with recombinant purified gD1 and produced high titers of antibody measured by enzyme-linked immunosorbent assay (ELISA; 1:5,120,000) as well as high plaque neutralization titer (1:320). Overall, the data indicated that stable expression in S2 cells is a practical way of synthesizing gD1 for use in structural and functional studies in the further study.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"42 1","pages":"384 - 391"},"PeriodicalIF":0.0,"publicationDate":"2016-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82515721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-01DOI: 10.1080/10826068.2015.1135466
Chaitali Vira, G. Prakash, J. Rathod, A. Lali
ABSTRACT Sedoheptulose 1,7-bisphosphatase (SBPase), a nuclear-encoded chloroplastic enzyme, is an important rate-limiting enzyme of the carbon fixation cycle (Calvin cycle). SBPase is unique to only photosynthetic organisms and is involved in the regeneration of ribulose-1,5-bisphosphate. SBPases from several sources have been studied for their induction and regulation. However, SBPase from Chlamydomonas reinhardtii CC-503, the widely studied model microalga, has not been isolated and functionally confirmed to date. In this study, the full-length cDNA for SBPase was isolated from C. reinhardtii CC-503 using anchored oligo(dT)24VGN primer for reverse transcription. The SBPase cDNA was cloned into pET28a expression vector for the production of 6X His-tagged protein in Escherichia coli BL21 (DE3) strain. Although initially most of the enzyme was obtained as insoluble protein aggregates, solubilization of protein was improved by optimization of protein induction with respect to growth temperature and isopropyl β-D-1-thiogalactopyranoside concentrations. The induced protein was purified by immobilized metal affinity chromatography using nickel–nitrilotriacetic acid resin in a phosphate-free buffer leading to an accurate SBPase activity measurement. The present study demonstrates, for the first time, successful cloning of C. reinhardtii CC-503 SBPase in E. coli leading to the expression of a functionally active enzyme.
{"title":"Cloning, expression, and purification of Chlamydomonas reinhardtii CC-503 sedoheptulose 1,7-bisphosphatase in Escherichia coli","authors":"Chaitali Vira, G. Prakash, J. Rathod, A. Lali","doi":"10.1080/10826068.2015.1135466","DOIUrl":"https://doi.org/10.1080/10826068.2015.1135466","url":null,"abstract":"ABSTRACT Sedoheptulose 1,7-bisphosphatase (SBPase), a nuclear-encoded chloroplastic enzyme, is an important rate-limiting enzyme of the carbon fixation cycle (Calvin cycle). SBPase is unique to only photosynthetic organisms and is involved in the regeneration of ribulose-1,5-bisphosphate. SBPases from several sources have been studied for their induction and regulation. However, SBPase from Chlamydomonas reinhardtii CC-503, the widely studied model microalga, has not been isolated and functionally confirmed to date. In this study, the full-length cDNA for SBPase was isolated from C. reinhardtii CC-503 using anchored oligo(dT)24VGN primer for reverse transcription. The SBPase cDNA was cloned into pET28a expression vector for the production of 6X His-tagged protein in Escherichia coli BL21 (DE3) strain. Although initially most of the enzyme was obtained as insoluble protein aggregates, solubilization of protein was improved by optimization of protein induction with respect to growth temperature and isopropyl β-D-1-thiogalactopyranoside concentrations. The induced protein was purified by immobilized metal affinity chromatography using nickel–nitrilotriacetic acid resin in a phosphate-free buffer leading to an accurate SBPase activity measurement. The present study demonstrates, for the first time, successful cloning of C. reinhardtii CC-503 SBPase in E. coli leading to the expression of a functionally active enzyme.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"9 1","pages":"810 - 814"},"PeriodicalIF":0.0,"publicationDate":"2016-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83890151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-01DOI: 10.1080/10826068.2015.1135463
Jianrong Wu, Shaozeng Lu, Z. Zheng, Li Zhu, X. Zhan
ABSTRACT A new protein derivatization method was developed with a block copolymer to reduce the immunogenicity of therapeutic proteins. The block copolymer consisted of polyethylene glycol (PEG) and polysialic acid (PSA), a nonimmunogenic and biodegradable biopolymer. Uricase was used as a model protein. Molecular weight analysis results indicated that the uricase–PEG–PSA conjugate was linked with 2.5 copolymers for each uricase unit. The residual enzyme activity of the uricase with modification by the PEG–PSA copolymer was 72.4%. The tolerance and stability to heat, acid, alkaline, and trypsin treatments significantly improved compared with the native uricase. The immunogenicity of uricase modified with PEG–PSA copolymer was remarkably reduced. The transmission electron microscopy results of the uricase–PEG–PSA conjugate showed a spherical hydrated shell with a larger particle size. These findings proved that the PSA–PEG–protein conjugate is a formulation that can potentially be used to deliver the protein and peptide-based drugs.
{"title":"Modification with polysialic acid–PEG copolymer as a new method for improving the therapeutic efficacy of proteins","authors":"Jianrong Wu, Shaozeng Lu, Z. Zheng, Li Zhu, X. Zhan","doi":"10.1080/10826068.2015.1135463","DOIUrl":"https://doi.org/10.1080/10826068.2015.1135463","url":null,"abstract":"ABSTRACT A new protein derivatization method was developed with a block copolymer to reduce the immunogenicity of therapeutic proteins. The block copolymer consisted of polyethylene glycol (PEG) and polysialic acid (PSA), a nonimmunogenic and biodegradable biopolymer. Uricase was used as a model protein. Molecular weight analysis results indicated that the uricase–PEG–PSA conjugate was linked with 2.5 copolymers for each uricase unit. The residual enzyme activity of the uricase with modification by the PEG–PSA copolymer was 72.4%. The tolerance and stability to heat, acid, alkaline, and trypsin treatments significantly improved compared with the native uricase. The immunogenicity of uricase modified with PEG–PSA copolymer was remarkably reduced. The transmission electron microscopy results of the uricase–PEG–PSA conjugate showed a spherical hydrated shell with a larger particle size. These findings proved that the PSA–PEG–protein conjugate is a formulation that can potentially be used to deliver the protein and peptide-based drugs.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"72 1","pages":"788 - 797"},"PeriodicalIF":0.0,"publicationDate":"2016-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85857621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-01DOI: 10.1080/10826068.2015.1135464
Jun Xia, Jiaxing Xu, Lei Hu, Xiaoyan Liu
ABSTRACT Poly(L-malic acid) (PMA) is a natural polyester with many attractive properties for biomedical application. However, the cost of PMA production is high when glucose is used as a carbon source. To solve this problem, cane molasses as a low-cost feedstock was applied for the production of PMA. Six pretreatment methods were applied to cane molasses before fermentation. Pretreatment with combined tricalcium phosphate, potassium ferrocyanide, and sulfuric acid (TPFSA) removed significant amounts of metal ions from cane molasses. The PMA concentration increased from 5.4 g/L (untreated molasses) to 36.9 g/L (TPFSA-pretreated molasses) after fermentation in shake flasks. A fed-batch fermentation strategy was then developed. In this method, TPFSA-pretreated cane molasses solution was continuously fed into the fermentor to maintain the total sugar concentration at 20 g/L. This technique generated approximately 95.4 g/L PMA with a productivity of 0.57 g/L/hr. The present study indicated that fed-batch fermentation using pretreated cane molasses is a feasible technique for producing high amounts of PMA.
{"title":"Enhanced poly(L-malic acid) production from pretreated cane molasses by Aureobasidium pullulans in fed-batch fermentation","authors":"Jun Xia, Jiaxing Xu, Lei Hu, Xiaoyan Liu","doi":"10.1080/10826068.2015.1135464","DOIUrl":"https://doi.org/10.1080/10826068.2015.1135464","url":null,"abstract":"ABSTRACT Poly(L-malic acid) (PMA) is a natural polyester with many attractive properties for biomedical application. However, the cost of PMA production is high when glucose is used as a carbon source. To solve this problem, cane molasses as a low-cost feedstock was applied for the production of PMA. Six pretreatment methods were applied to cane molasses before fermentation. Pretreatment with combined tricalcium phosphate, potassium ferrocyanide, and sulfuric acid (TPFSA) removed significant amounts of metal ions from cane molasses. The PMA concentration increased from 5.4 g/L (untreated molasses) to 36.9 g/L (TPFSA-pretreated molasses) after fermentation in shake flasks. A fed-batch fermentation strategy was then developed. In this method, TPFSA-pretreated cane molasses solution was continuously fed into the fermentor to maintain the total sugar concentration at 20 g/L. This technique generated approximately 95.4 g/L PMA with a productivity of 0.57 g/L/hr. The present study indicated that fed-batch fermentation using pretreated cane molasses is a feasible technique for producing high amounts of PMA.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"44 1","pages":"798 - 802"},"PeriodicalIF":0.0,"publicationDate":"2016-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86491401","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2016-02-01DOI: 10.1080/10826068.2015.1135465
J. Rathod, G. Prakash, Chaitali Vira, A. Lali
ABSTRACT Parachlorella kessleri is a promising oil-bearing marine alga which shows decreased growth under high light stress. Osmolytes are known to relieve stress by protecting the cell membrane, proteins, and enzymes. Enhanced production of osmolyte (trehalose) was thus used to relieve stress in P. kessleri by overexpression of trehalose phosphate synthase (TPS) gene. Transformed P. kessleri was grown under different light regimes to study the effect of trehalose overproduction on growth. Study of one of the TPS transformants showed increased trehalose as well as increased biomass and decreased pigments, reactive oxygen species, and lipid peroxidation of cell membrane. The improved photosynthetic performance of the transformant was also signified by pulse-amplitude-modulated fluorometric analysis. All of these factors reveal improved stress tolerance under high light conditions by increased trehalose accumulation due to TPS overexpression in P. kessleri.
{"title":"Trehalose phosphate synthase overexpression in Parachlorella kessleri improves growth and photosynthetic performance under high light conditions","authors":"J. Rathod, G. Prakash, Chaitali Vira, A. Lali","doi":"10.1080/10826068.2015.1135465","DOIUrl":"https://doi.org/10.1080/10826068.2015.1135465","url":null,"abstract":"ABSTRACT Parachlorella kessleri is a promising oil-bearing marine alga which shows decreased growth under high light stress. Osmolytes are known to relieve stress by protecting the cell membrane, proteins, and enzymes. Enhanced production of osmolyte (trehalose) was thus used to relieve stress in P. kessleri by overexpression of trehalose phosphate synthase (TPS) gene. Transformed P. kessleri was grown under different light regimes to study the effect of trehalose overproduction on growth. Study of one of the TPS transformants showed increased trehalose as well as increased biomass and decreased pigments, reactive oxygen species, and lipid peroxidation of cell membrane. The improved photosynthetic performance of the transformant was also signified by pulse-amplitude-modulated fluorometric analysis. All of these factors reveal improved stress tolerance under high light conditions by increased trehalose accumulation due to TPS overexpression in P. kessleri.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"27 1","pages":"803 - 809"},"PeriodicalIF":0.0,"publicationDate":"2016-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75738880","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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