Enhanced recombinant protein capture, purity and yield from crude bacterial cell extracts by N-Lauroylsarcosine-assisted affinity chromatography.

BACKGROUND: Recombinant proteins cover a wide range of biomedical, biotechnological, and industrial needs. Although there are diverse available protocols for their purification from cell extracts or from culture media, many proteins of interest such as those containing cationic domains are difficult to purify, a fact that results in low yields of the final functional product. Unfortunately, this issue prevents the further development and industrial or clinical application of these otherwise interesting products. RESULTS: Aiming at improving the purification of such difficult proteins, a novel procedure has been developed based on supplementing crude cell extracts with non-denaturing concentrations of the anionic detergent N-Lauroylsarcosine. The incorporation of this simple step in the downstream pipeline results in a substantial improvement of the protein capture by affinity chromatography, an increase of protein purity and an enhancement of the overall process yield, being the detergent not detectable in the final product. CONCLUSION: By taking this approach, which represents a smart repurposing of N-Lauroylsarcosine applied to protein downstream, the biological activity of the protein is not affected. Being technologically simple, the N-Lauroylsarcosine-assisted protein purification might represent a critical improvement in recombinant protein production with wide applicability, thus smothering the incorporation of promising proteins into the protein market.

Low-cost modular chromatography column rack and vial holders.

Gravity-driven chromatography columns are used in scientific, engineering, medical, and industrial fields to separate desired compounds from solutions. Running multiple columns simultaneously saves time and improves procedural consistency. Though column chromatography is widely used, to meet their laboratory needs many investigators must resort to designing and fabricating custom racks for holding their chromatography columns. We have created a robust column rack design, with collection vial holders, that is easily made, inexpensive to build, and may be easily adapted to fit experimental needs. The column holder can be made to hold various sizes of columns (and can be interchanged as necessary); the height of columns above collection vials can be precisely set; and the design is modular, so the rack and vial holders can be expanded to accommodate the desired numbers of columns and the numbers and sizes of vials used to collect fractions eluted from each column. Importantly, the rack is made of inexpensive, readily-available materials and the fabrication is straightforward. Here we present details of the rack's features, a list of materials, and instructions for making it. We hope our design will help investigators who perform gravity-driven column chromatography.

Comparative Analysis of Four Complete Mitochondrial Genomes of Epinephelidae (Perciformes).

Groupers are commercial, mainly reef-associated fishes, classified in the family Epinephelidae (Perciformes). This study first sequenced the complete mitogenomes of Cephalopholis leopardus, Cephalopholis spiloparaea, Epinephelus amblycephalus, and Epinephelus hexagonatus. The lengths of the four Epinephelidae mitogenomes ranged from 16,585 base pair (bp) to 16,872 bp with the typical gene order. All tRNA genes had a typical cloverleaf structure, except the tRNA-Ser (AGY) gene which was lacking the entire dihydrouridine arm. The ratio of nonsynonymous substitution (Ka) and synonymous substitution (Ks) indicated that four groupers were suffering a purifying selection. Phylogenetic relationships were reconstructed by Bayesian inference (BI) and maximum likelihood (ML) methods based on all mitogenomic data of 41 groupers and 2 outgroups. The identical topologies result with high support values showed that Cephalopholis and Epinephelus are not monophyletic genera. Anyperodon and Cromileptes clustered to Epinephelus. Aethaloperca rogaa and Cephalopholis argus assembled a clad. Cephalopholis leopardus, C. spiloparaea, and Cephalopholis miniata are also in a clade. Epinephelushexagonatus is close to Epinephelus tauvina and Epinephelus merra, and E. amblycephalus is a sister group with Epinephelus stictus. More mitogenomic data from Epinephelidae species are essential to understand its taxonomic status with the family Serranidae.

Antibody persistence upto 5 years after primary immunization and booster with an inactivated chromatographically purified Vero cell-derived Japanese encephalitis vaccine in Thai children.

Japanese encephalitis is the main cause of viral encephalitis in Asia. In a previous single-arm vaccine trial, an inactivated chromatographically purified Japanese encephalitis Vero cell vaccine (CVI-JE; JEVACTM) was safe and immunogenic in 152 Thai children aged 1-3 years receiving a 2-dose primary immunization and booster dose 1 year later. We conducted a 5-year follow-up assessment of the persistence of the immune response the 144 children remaining in this cohort after first booster dose. Immunity was assessed by 50% plaque reduction neutralization test annually for up to 5 years post-booster. Seroprotection rates (95%CI) decreased from 100% (97.1-100) at 1 year post-booster to 93% (85.0-98.3) at 5 years post-booster. No serious vaccine-related adverse events or Japanese encephalitis infections were reported. A 2-dose primary immunization and booster 1 year later with CVI-JE provided long-lasting immunity in the majority of children.

Construction of hierarchical 2D/2D Ti3C2/MoS2 nanocomposites for high-efficiency solar steam generation.

Solar steam generation has been considered one of the most promising approaches for dealing with the energy and freshwater resource crises in recent years. However, achieving high efficiency in photo-thermal conversion remains a considerable challenge. Here, a series of hierarchical Ti3C2/MoS2 nanocomposites were designed for steam generation by a hydrothermal method. When the mass fraction of MoS2 reached 65 wt% (TM-3), the Ti3C2/MoS2 nanocomposite presented a strong broad-band light absorption of 92.4% from the UV to NIR region because of the accordion-like layered structure. The evaporation rate and solar-thermal conversion efficiency of the TM-3 with as-fabricated evaporator could reach 1.36 kg·m-2·h-1 and 87.2% under 1 kW/m2, due to the excellent light absorption ability of TM-3 and the low thermal energy loss (8.8%) of the evaporator. Meanwhile, TM-3 permits the evaporator to have remarkable cycle stability because of its hydrophobic properties. Moreover, TM-3 showed excellent seawater desalination and wastewater treatment abilities. Thus, the excellent light absorption ability, photo-thermal conversion efficiency, and stability of the overall system suggested that these nanocomposites show great potential applications in synergetic solar desalination and sewage treatment.

[Purification and characterization of two PR-10 protein isoforms from the crude drug of Angelica sinensis].

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²âº had an activating effect on the ribonuclease activities of two isoforms while Ca²âº, Mg²âº, Zn²âº, Mn²âº, Ag⁺, Cu²âº, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.

Purification and characterization of two PR-10 protein isoforms from the crude drug of Angelica sinensis / 生物工程学报

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.

Immunogenicity and safety of inactivated chromatographically purified Vero cell-derived Japanese encephalitis vaccine in Thai children.

Inactivated mouse-brain-derived Japanese encephalitis vaccine has a worrisome safety profile and the live attenuated vaccine is unsuitable in immunodeficiency. This study aimed to evaluate the immunogenicity and safety of an inactivated chromatographically purified Vero-cell-derived JE vaccine (CVI-JE, Beijing P-3 strain) in children. 152 healthy Thai children, with an average (SD) age of 14.4 (3.8) months, received 3 doses of CVI-JE on days 0, 7-28, and one year. Homologous JE neutralizing antibody titers (NT) were measured. All subjects had seroprotection [geometric mean titer (GMT) 150] 28 days' post 2nd vaccination. The seroprotection rates at 1 year after primary series and and 1 month after the booster were 89.3% (GMT 49) and 100% (GMT 621), respectively. Local and systemic reactions-fever (17.6%), vomiting (8%), and poor appetite (5.3%)-were noted within 28 days' post-vaccination. All these symptoms were self-limited. CONCLUSIONS: CVI-JE is safe, immunogenic, and provided high NT.

Expression and purification of human placental growth factor 2 using the Pichia pastoris expression system / 中国药科大学学报

To prepare the human placental growth factor 2 (PIGF-2 ),human PIGF-2 gene was cloned into pPIC9K vector to construct the recombinant pPIC9K-PIGF-2 vector. Linearized recombinant pPIC9K-PIGF-2 was transformed into Pichia pastoris by electroporation. YPD-Geneticin plate was used to screen geneticin hyper-resistant colonies. The positive colonies were verified by PCR. Results of SDS-PAGE and Western blot showed that recom-binant human PIGF-2 was expressed after being induced by methanol. Using its characteristic heparin binding, recombinant human PIGF-2 was successfully purified by heparin affinity column chromatography.

Study on the techniques of purification flavones from Gnaphalium japonicum Thunb. by D-101 macroporous adsorption resin / 国际中医中药杂志

Objective To investigate the optimal isolation and purification conditions of flavonoids in Gnaphlium affine Thunb. and the content of flavonoids in different parts of Gnaphlium affine Thunb. was measured. Methods The separation and purification abilities of D-101 macroporoue adsorbing resins for flavonoids in Gnaphlium affine Thunb. were studied with adorption and desorption as index. The static adsorption and dynamic adsorption methods were used to analyze the effects of static saturated adsorption, static elution rate, sample concentration, sample pH value, eluent concentration and amount of eluent. The flavonoids concentrations were determined with rutin as standard. Results The D-101 macroporous adsorption resin had good effect on the separation and purification of flavonoids from Gnaphlium affine Thunb.; The optimal conditions for purification were: sample concentration 1.0 mg/ml, sample pH=4, 60％ ethanol as desorption solvent, washing flow 2 BV/h, with these parameters. With such condition, the purity of flavonoids in Gnaphlium affine Thunb. was 60.92％; The content of flavonoids in Gnaphlium affine Thunb. showed the highest content was in the stem, the second in the flower, and the least in the root. Conclusions The purification of flavonoids from the D-101 macroporous adsorption resin increased in the best purified separation conditions and the content of flavonoids was the highest in the leaf.

[Response surface optimization of purification process for pigment from Coreopsis tinctoria by macroporous resins].

To optimize the purification process of pigments from Coreopsis tinctoria with macroporous resins by establishing second regression model with response surface methodology. The experiment showed that XDA-7 resin had the best purification effect for pigments from C. tinctoria. The optimal absorption conditions for pigments from C. tinctoria were determined as follows： concentration of pigments solution 2.7 g•L⁻¹, flow rate 6 mL•min⁻¹, pH 6. Under these conditions, the absorption rate of pigments was up to 94.16%. Optimal desorption conditions were as follows： ethanol concentration 64%, flow rate 5 mL•min⁻¹, elution dosage 4 BV. Under these conditions, pigment desorption rate was as high as 98.72%.

Response surface optimization of purification process for pigment from Coreopsis tinctoria by macroporous resins / 中国中药杂志

To optimize the purification process of pigments from Coreopsis tinctoria with macroporous resins by establishing second regression model with response surface methodology. The experiment showed that XDA-7 resin had the best purification effect for pigments from C. tinctoria. The optimal absorption conditions for pigments from C. tinctoria were determined as follows： concentration of pigments solution 2.7 g•L⁻¹, flow rate 6 mL•min⁻¹, pH 6. Under these conditions, the absorption rate of pigments was up to 94.16%. Optimal desorption conditions were as follows： ethanol concentration 64%, flow rate 5 mL•min⁻¹, elution dosage 4 BV. Under these conditions, pigment desorption rate was as high as 98.72%.

A study on proliferation and behavior of retinal pigment epithelial cells on purified alginate films.

BACKGROUND AND OBJECTIVES: Alginate, an anionic polysaccharide distributed widely in the cell walls of brown algae, is used in biomedical applications. However, alginate' s performance as a biomaterial, has limited by its several contamination such as endotoxins, proteins and polyphenols. METHODS AND RESULTS: To overcome this problem, we have developed using modified Korbutt method for alginate purification. After purification, we made alginate films and used for retinal pigment epithelial cell (RPEs) regeneration. ARPE-19 cells were seeded in non-purified and purified alginate films, and then cell viability and proliferation were estimated by MTT assay and RT-PCR was performed to assess specific cell expression. ARPE-19 cell-loaded alginate films were evaluated specific protein expression by through AEC staining and we examined the cell adhesion by scanning electron micro scopy (SEM). CONCLUSIONS: In this result, ARPE-19 cells in purified alginate films had higher cell proliferative rate and phenotypic expression than those on non-purified alginate films. The results suggest that purified alginate is useful for RPEs regeneration.

Purification of monoclonal antibody against PGRP and its activity for small cell lung cancer in vitro / 肿瘤研究与临床

Objective To explore the effect of purification on monoclonal antibody (MAb) against PGRP by Protein A-Sepharose affinity chromatography, and to provide some based data for the purification of other antibody using the same method. Methods The ascites which include MAb was purified by Protein A-Sepharose affinity chromatography. The purity and activity of MAb was tested by SDS-PAGE and ELISA. The biological function was identified by flow cytometer and immunohistochemistry. Results The average concentration of protein in ascites before purification is 23.62 mg/ml. Before and after purification, the total protein is 148.79 mg and 146.67 mg, respectively. The recovery coefficient of protein is 98.58%. The concentration of MAb in ascites is 5.21 mg/ml averagely. The MAb purity is more than 95 %. The immunoactivity of purified antibody is higher than that of unpurified antibody. Conclusion The purity of MAb against PGRP purified by Protein A-Sepharose affinity chromatography is very high. The immunoactivity of purified antibody is higher than that of unpurified antibody. So the ProteinA-Sepharose affinity chromatography is a rapid, convenient and reliable method for the purification of MAb Against PGRP.

Expression and Purification of the Fusion Protein Anti-epilepsy Peptide/Glutathiones-transferase / 浙江中医药大学学报

[Aim] To gain the fusion protein purified GST-AEP.[Methods] The protein GST-AEP was expressed in E.Coli-DH5? as a fusion protein induced by IPTG.The protein was a kind of inclusion body.The purifying and refolding to inclusion body were optimized.The purity of GST-AEP was identified by 12% SDS-PAGE and thin-layer scanning analysis.The quantitation of the fusion protein GST-AEP was done with BCA Protein Assay.[Results] Purity of GST-AEP was higher than 90% and concentration was about 0.163?g/?l.[Conclusion] The fusion protein was highly purified and the method of fusion protein purification from the inclusion body was developed,which was the basis for further study on AEP.

THE ISOLATION, PURIFICATION AND IDENTIFICATION OF WISTAR RAT'S ISLET / 解剖学报

Objective The experiment aims at probing the best condition of the isolation and purification of rat islets.Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation.Then the purified islets were subjected to histological staining,electron microscopy and radioimmunoassay for identification of specificity and viability.Results The histological staining revealed that the viability and the purity of the purified islets were above 95％and 85％respectively.Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules.Radioimmunoassay demonstrated the secreted insulin concerntration between low-glucose groups and high-glucose groups varied significantly,which verified the good function of the islets.Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion.There aye many factors that influence the quantity and quality of the acquired islets,such as the completed expansion of pancreas,the concentration and viability of collagenase and the digested time,etc.

The Purification of Astrocytes From Cerebral Cortex of SD Rats / 医学研究杂志

Objective To simply the method of how to obtain cultures rich in astrocytes from SD rat cerebral cortex which could be utilized in vitro experiments.Methods The neonatal rat cerebral cortex was made into suspension by mechanical dissociation,and then reduced other cells by differential velocity adherent technique,shaking in orbital shaker and passage of cultured cells.After purification,the cultured cells were identified by double immunofluorescence staining and SABC.Results We successfully obtain cultures rich in astrocytes and the proportion of astrocytes were more than 95%.Conclusion The method described above was reliable in obtaining the high purity astrocytes from neonatal rat cerebral cortex and double immunofluorescence staining was more vivid and direct.

The purification and identification of phospholipid-binding anticoagulation protein from agkistrodon halys brevicaudus venom / 中国药理学通报

Aim To purify Phospholipid-binding anticoagulation protein(PBAP) from Agkistrodon halys Brevicaudus Venom and study the biochemical characterization.Methods The Phospholipid-binding anticoagulation protein was purified from Agkistrodon halys Brevicaudus Venom by Cation ion exchange chromatography on CM Sephadex C-25 and negative ion exchange chromatography on DEAE Sepharose CL-6B,gel filtration on Sephacryl S-200 and Sephadex G-75 chromatography.Its anticoagulant activities in vitro were assayed by activated partial thromboplastin time(APTT);its molecular weight was calculated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and its isoelectric point was estimated by the isoelectric focusing electrophoresis.Binding experiments of anticoagulation protein to phospholipids vesicles were performed with thinlayer chromatography.Results A kind of protein was purified from Agkistrodon halys Brevicaudus Venom which was able to prolong APTT.The SDSPAGE showed that it was dimer and its molecular weight was 24.0?10~3 under non-reducing condition and 14.6?10~3 under reducing condition.The isoelectric point was pH 5.2 by the isoelectric focusing electrophoresis.Having arginine ester-hydrolyzing enzyme and binding Phospholipid activify,its effect on APTT was activity stronger with concentration increasing.Conclusion It is a successful method of the purification of Phospholipid-binding anticoagulation protein from the Agkistrodon halys Brevicaudus Venom.

Advances in Gene-engineering Production of Defensins / 微生物学通报

This paper reviews the recent advances in recombinant expression and purification of defensins, including the choice of host cells, vectors and expression strategies in prokaryotic and eukaryotic cell ex- pression systems, as well as the status of purification processes. By summarizing the problems existed in the production and clinical applications of defensins, the authors here also pointed out the research directions for defensins, and conceived the prospects for its exploitation in the future.