Preparative Biochemistry and Biotechnology最新文献_第7页

Characterization of recombinant human granulocyte colony-stimulating factor expression by FT-IR spectroscopy: Studies on thermal induction and media formulation on the stability of the protein secondary structure 重组人粒细胞集落刺激因子表达的FT-IR表征:热诱导及培养基配方对蛋白二级结构稳定性的影响

Preparative Biochemistry and Biotechnology Pub Date : 2016-08-17 DOI: 10.1080/10826068.2015.1084933

Sandeep R Vemula, Sushma Vemula, A. Dedaniya, Rajesh Kumar Kante, Srinivasa Reddy Ronda

{"title":"Characterization of recombinant human granulocyte colony-stimulating factor expression by FT-IR spectroscopy: Studies on thermal induction and media formulation on the stability of the protein secondary structure","authors":"Sandeep R Vemula, Sushma Vemula, A. Dedaniya, Rajesh Kumar Kante, Srinivasa Reddy Ronda","doi":"10.1080/10826068.2015.1084933","DOIUrl":"https://doi.org/10.1080/10826068.2015.1084933","url":null,"abstract":"ABSTRACT The Fourier-transform infrared (FT-IR) spectroscopic approach has been employed to understand the recombinant human G-CSF (rhG-CSF) protein accumulation, secondary structure, and thermal stability in Escherichia coli grown under a temperature shift strategy (37 and 28°C) in various media formulations. The choline + sodium pyruvate (37°C) and sodium pyruvate (28°C) formulations have shown the highest inclusion body (IB) accumulation of 0.41 and 0.46 mg/mL, respectively. Furthermore, insights on the structure of the rhG-CSF within IBs and intact cells have been investigated through secondary structure analysis. Thermal stability experiments were also carried out to explain the pattern of the second derivative structure of rhG-CSF. The studies showed that choline + sodium pyruvate formulation has preserved the protein secondary structure even at 82°C. Overall, the FT-IR spectroscopic technique can also be adopted to accelerate the characterization of other recombinant therapeutic proteins of E. coli origin.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84221697","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}

引用次数: 0

Optimization of submerged fermentation medium for citrinin-free monascin production by Monascus 红曲霉生产无柑桔素红曲霉素深层发酵培养基的优化

Preparative Biochemistry and Biotechnology Pub Date : 2016-03-07 DOI: 10.1080/10826068.2015.1135461

Di Chen, Yuan Xue, Mian-hua Chen, Zhenjing Li, Chang-lu Wang

引用次数: 22

Solanum melongena polyphenol oxidase biosensor for the electrochemical analysis of paracetamol 龙葵多酚氧化酶生物传感器对乙酰氨基酚的电化学分析

Preparative Biochemistry and Biotechnology Pub Date : 2016-03-01 DOI: 10.1080/10826068.2016.1155060

L. F. Garcia, Stephen Rathinaraj Benjamin, R. Antunes, F. M. Lopes, V. Somerset, E. Gil

{"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":null,"pages":null},"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}

引用次数: 19

Structure modeling and functional analysis of recombinant dextransucrase from Weissella confusa Cab3 expressed in Lactococcus lactis 乳酸菌重组葡聚糖酶Cab3的结构建模及功能分析

Preparative Biochemistry and Biotechnology Pub Date : 2016-02-09 DOI: 10.1080/10826068.2016.1141299

S. Shukla, A. Verma, I. Kajala, Antti Nyyssolä, Rwivoo Baruah, K. Katina, R. Juvonen, M. Tenkanen, A. Goyal

{"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":null,"pages":null},"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}

引用次数: 10

Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia 克级生产的质粒pUDK-HGF与目前良好的生产规范，用于基因治疗严重肢体缺血

Preparative Biochemistry and Biotechnology Pub Date : 2016-02-06 DOI: 10.1080/10826068.2016.1141302

Chunsheng Hu, Xiaochen Cheng, Yuxin Lu, Zu-ze Wu, Qingling Zhang

{"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":null,"pages":null},"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}

引用次数: 0

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems 双水相-胶束系统液液萃取抗菌肽P34

Preparative Biochemistry and Biotechnology Pub Date : 2016-02-06 DOI: 10.1080/10826068.2016.1141301

V. Sant'anna, Ana Paula Folmer Correa, Amanda de Souza da Motta, A. Brandelli

{"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":null,"pages":null},"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}

引用次数: 8

Expression, purification, and characterization of recombinant human H-chain ferritin 重组人h链铁蛋白的表达、纯化和表征

Preparative Biochemistry and Biotechnology Pub Date : 2016-02-06 DOI: 10.1080/10826068.2016.1141300

Wenyan Zou, Xiaoyu Liu, Dian-hua Chen, Jie Wang, Xi Zhao, Jiahuang Li, Lina Ji, Z. Hua

引用次数: 5

Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells 单纯疱疹病毒1型重组糖蛋白D在果蝇S2细胞中的表达及免疫原性

Preparative Biochemistry and Biotechnology Pub Date : 2016-02-02 DOI: 10.1080/10826068.2015.1045610

Hongyan Mao, Xiaofei Zhao, Hongjuan Zhu, Jingxia Guo, Zhenghai Ma

{"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":null,"pages":null},"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}

引用次数: 2

Cloning, expression, and purification of Chlamydomonas reinhardtii CC-503 sedoheptulose 1,7-bisphosphatase in Escherichia coli 莱茵衣藻CC-503 sedoheptulose 1,7-双磷酸酶在大肠杆菌中的克隆、表达和纯化

Preparative Biochemistry and Biotechnology Pub Date : 2016-02-01 DOI: 10.1080/10826068.2015.1135466

Chaitali Vira, G. Prakash, J. Rathod, A. Lali

{"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":null,"pages":null},"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}

引用次数: 3

Modification with polysialic acid–PEG copolymer as a new method for improving the therapeutic efficacy of proteins 聚唾液酸-聚乙二醇共聚物修饰是提高蛋白质治疗效果的新方法

Preparative Biochemistry and Biotechnology Pub Date : 2016-02-01 DOI: 10.1080/10826068.2015.1135463

Jianrong Wu, Shaozeng Lu, Z. Zheng, Li Zhu, X. Zhan

引用次数: 5