Recovery of thrombopoietin during purification

A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) was previously purified by a six-step purification procedure. However, the exact quantity of TSF that was recovered, through the various purification procedures, was unknown because of the absence of a method for establishing a unit of measure of TSF. In the present work dose-response relationships on both the crude TSF preparations and on the more highly purified TSF were determined. TSF units were calculated from the dose-response curves. A unit of TSF is defined as the amount of material (mg) that is required to increase the percentages 35S incorporation into platelets of immunothrombocythemic mice by 50% above the baseline. The results of determining the TSF units on the crude TSF preparation indicated that 0.11 unit (U) of TSF/mg protein was present. Results showed that the specific activity of TSF can be increased to about 3.6 U/mg by a single purification procedure using Sephadex G-75 column chromatography. Increased specific activities were obtained by additional purification steps, i.e., DEAE-cellulose column chromatography, SE-HPLC, DEAE-HPLC, and SDS-PAGE. The purified product appears to have a specific activity of about 11,000 U/mg of protein with 0.00003% of the protein and 1.1% of the TSF recovered from the starting material. Establishing a unit of measure for TSF will allow calculations of its degree of purity, provide a method for quantitation of recoveries of activities after various purification procedures, and allow comparisons of results from different experiments and different laboratories.     

Recovery and purification of plant-made recombinant proteins

Plants are becoming commercially acceptable for recombinant protein production for human therapeutics, vaccine antigens, industrial enzymes, and nutraceuticals. Recently, significant advances in expression, protein glycosylation, and gene-to-product development time have been achieved. Safety and regulatory concerns for open-field production systems have also been addressed by using contained systems to grow transgenic plants. However, using contained systems eliminates several advantages of open-field production, such as inexpensive upstream production and scale-up costs. Upstream technological achievements have not been matched by downstream processing advancements. In the past 10 years, the most research progress was achieved in the areas of extraction and pretreatment. Extraction conditions have been optimized for numerous proteins on a case-by-case basis leading to the development of platform-dependent approaches. Pretreatment advances were made after realizing that plant extracts and homogenates have unique compositions that require distinct conditioning prior to purification. However, scientists have relied on purification methods developed for other protein production hosts with modest investments in developing novel plant purification tools. Recently, non-chromatographic purification methods, such as aqueous two-phase partitioning and membrane filtration, have been evaluated as low-cost purification alternatives to packed-bed adsorption. This paper reviews seed, leafy, and bioreactor-based platforms, highlights strategies for the primary recovery and purification of recombinant proteins, and compares process economics between systems. Lastly, the future direction and research needs for developing economically competitive recombinant proteins with commercial potential are discussed.     

Technoeconomic analysis for biofuels and bioproducts

1. Download : Download high-res image (160KB)
2. Download : Download full-size image

     

生物制品研制开发动态

本概述了近年来生物制品研制领域的新动态,包括１９９９年我国新药研制相关法规的修订、有军事背景的生物制品的开发和研制,及在生物药物研制中值得重视的新生物技术领域。另外,介绍了生物制品的知识产权保护情况及如何查找生物技术专利资源。     

Ascaris suum: aldolase I purification and immunologic study

Fructose-1,6-diphosphate d-glyceraldehyde-3-phosphate lyase (aldolase) (EC 4.1.2.13) from the body wall of Ascaris suum was purified 80-fold by a combination of salt precipitation and ion-exchange chromatography. A group of pigs was immunized with the purified aldolase preparation and was subsequently challenged with infective Ascaris larvae. The immunized animals showed clinical and histopathologic symptoms of acute sensitization reaction. Thrice as many larvae were found in the nonimmunized control pigs as compared to the immunized animals.     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.Key words: monoclonal antibody, recovery, purification, chromatography, membrane, filtration, platform process     

Recovery and purification process development for monoclonal antibody production

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.     

Leishmanicidal activity of fungal bioproducts: A systematic review

The genome mining of biosynthetic genes from fungi demonstrates the enormous pharmacological potential that is still little explored. These results have encouraged the scientific community to invest in fungi as a source of innovative alternatives for the treatment of neglected diseases, such as leishmaniasis. Therefore, this work aimed to identify, through a systematic search in the databases of PubMed, Lilacs and Scielo, the existing evidence in the literature regarding the efficacy of the leishmanicidal activity of fungal bioproducts that represent new starting points for the advancement of pharmacotherapy of leishmaniasis. During the search process, 59 articles met all the eligibility criteria and, therefore, were included in this review. The studies demonstrate that different prospecting, cultivation, biotechnological and synthetic modification strategies contribute to the discovery and development of new therapeutic fungal compounds. 39 (66.1%) of the studies presented at least one isolated compound with leishmanicidal activity, while 20 (33.9%) evaluated only crude extracts or semipurified fractions. Terpenes, steroids and quinones were the most prevalent chemical classes among the isolated compounds. There are many studies about active compounds that have been isolated from Penicillium and Aspergillus genera. A large majority (89.8%) of the selected studies been conducted in vitro. Only six studies performed in vivo assay. The species of Leishmania amazonensis and Leishmania donovani were the most evaluated. The results support the hypothesis of the pharmacological potential of fungal bioproducts in the treatment of leishmaniasis.     

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.

     

Recovery and purification of highly aggregation-prone disulfide-containing peptides: application to islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a 37-residue pancreatic hormone. It is responsible for the formation of islet amyloid in vivo and is very insoluble and aggregation-prone in vitro, particularly at basic pH. The peptide contains a disulfide bridge between residues two and seven and an amidated C terminus. There is no reported expression system for the production of amidated IAPP. The peptide is difficult to synthesize and formation of the disulfide by traditional methods is problematic. We have found that the use of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) or dimethyl sulfoxide (DMSO) significantly improves disulfide formation and purification of highly aggregation-prone IAPP sequences. The use of these organic solvents increases the solubility of the hydrophobic peptides, avoids the use of aqueous basic solutions, and eliminates the need for continuous stirring during oxidation to form the Cys-2 to Cys-7 disulfide bridge. Elimination of the stirring step and basic solution helps to reduce aggregation and allows for more consistent high-performance liquid chromatography (HPLC) retention times. Formation of the intramolecular disulfide using DMSO was found to be the most effective method for IAPP oxidation, reducing the reaction time from 24 to 5 h. Aggregated IAPP can be resolubilized by HFIP or DMSO and recovered by HPLC with very good yield.     

Casein kinase I in bovine sperm: purification and characterization

A highly purified preparation of sperm casein kinase I was obtained by sequential chromatography with phosphocellulose, gel filtration on sephacryl S-300, Affi-gel blue and DEAE-Cellulose. The chromatographic behavior and properties of the enzyme suggest that the sperm enzyme is similar to casein kinase I from other tissues. Antibodies against calf thymus casein kinase I cross-react with the sperm enzyme. A special feature of the sperm enzyme is that the activity is stimulated by spermine.     

Drosophila topoisomerase I: isolation, purification and characterization.

We have purified and characterized topoisomerase I from Drosophila melanogaster. The molecular weight of the enzyme is 135,000; 100,000, 90,000, and 65,000 molecular weight products result from degradation of the enzyme. The enzyme relaxes both positive and negative supercoiled DNA. Mg++ is not absolutely required, but stimulates the enzymatic activity considerably.     

Human liver GTP cyclohydrolase I: purification and some properties

Human liver guanosine triphosphate (GTP) cyclohydrolase I has been purified more than 1,700-fold to what appears to be homogeneity. The active enzyme complex has an estimated molecular weight of 453,000 +/- 11,500 by gel filtration chromatography. It consists of a polypeptide of 149,000 +/- 4,000 mol wt by SDS-polyacrylamide gel electrophoresis. The activity of the enzyme is heat stable and is inhibited by di- and trivalent cations. The enzyme has an optimum pH of 7.7 in sodium phosphate buffer. It uses GTP as a sole substrate, with a Km of 116 microM.     

Multifaceted roles of duckweed in aquatic phytoremediation and bioproducts synthesis

Duckweed (Lemnaceae) is a fast‐growing aquatic vascular plant. It has drawn an increasing attention worldwide due to its application in value‐added nutritional products and in sewage disposal. In particular, duckweed is a promising feedstock for bioenergy production. In this review, we summarized applications of duckweed from the following four aspects. Firstly, duckweed could utilize nitrogen, phosphorus, and inorganic nutrition in wastewater and reduces water eutrophication efficiently. During these processes, microorganisms play an important role in promoting duckweed growth and improving its tolerance to stresses. We also introduced our pilot‐scale test using duckweed for wastewater treatment and biomass production simultaneously. Secondly, its capability of fast accumulation of large amounts of starch makes duckweed a promising bioenergy feedstock, catering the currently increasing demand for bioethanol production. Pretreatment conditions prior to fermentation can be optimized to improve the conversion efficiency from starch to bioethanol. Furthermore, duckweed serves as an ideal source for food supply or animal feed because the composition of amino acids in duckweed is similar to that of whey protein, which is easily digested and assimilated by human and other animals. Finally, severing as a natural plant factory, duckweed has shown great potential in the production of pharmaceuticals and dietary supplements. With the surge of omics data and the development of Clustered Regularly Interspaced Short Palindromic Repeats technology, remodeling of the metabolic pathway in duckweed for synthetic biology study will be attainable in the future.     

Plant synthetic biology innovations for biofuels and bioproducts

Plant-based biosynthesis of fuels, chemicals, and materials promotes environmental sustainability, which includes decreases in greenhouse gas emissions, water pollution, and loss of biodiversity. Advances in plant synthetic biology (synbio) should improve precision and efficacy of genetic engineering for sustainability. Applicable synbio innovations include genome editing, gene circuit design, synthetic promoter development, gene stacking technologies, and the design of environmental sensors. Moreover, recent advancements in developing spatially resolved and single-cell omics contribute to the discovery and characterization of cell-type-specific mechanisms and spatiotemporal gene regulations in distinct plant tissues for the expression of cell- and tissue-specific genes, resulting in improved bioproduction. This review highlights recent plant synbio progress and new single-cell molecular profiling towards sustainable biofuel and biomaterial production.     

Partial purification and characterization of locust allatotropin I

Methanol extracts of locust brains, corpora cardiaca (CC), and suboesophageal ganglia (SOG) were separated by gradient and/or isocratic reverse-phase high-performance liquid chromatography (HPLC) and allatotropic activity monitored in the eluted fractions. A major peak of activity, separated by isocratic separation with 12% 2-propanol, designated allatotropin I, exhibited identical retention times in the three tissue extracts. Doseresponse curves of allatotropin I indicate similar content in brain and CC-equivalents, whereas optic lobes, similarly separated by isocratic HPLC, contain only one-tenth of this amount of allatotropin. Allatotropin I is resistant to boiling and is susceptible to tryptic and chymotryptic digestion. Methanol extracts of thoracic muscle, Malpighian tubules, fat body or ovaries, similarly prepared and boiled, did not exhibit allatotropic activity at high doses of tissue equivalents.     

Acetohydroxy acid synthase I of Escherichia coli: purification and properties.

Several properties of the three acetohydroxy acid synthases of Escherichia coli have been compared in crude extracts. The three enzymes can be readily distinguished from each other. Acetohydroxy acid synthase I, the product of the ilvB gene, has been purified to near homogeneity. The purification was made possible by the fact that the enzyme was maintained in buffers of a high ionic strength or in buffers containing glycerol. Density gradient centrifugation studies indicated that the enzyme exists as a dimer of subunits of similar (60,000) molecular weight in buffers containing glycerol with or without two of the cofactors. Mg2+ and thiamine diphosphate. When flavine adenine dinucleotide was added along with Mg2+ and thiamine diphosphate, an increase in the rate of sedimentation occurred that was thought to be due to a rapid tetramer-dimer interconversion. The addition of pyruvate, the substrate, along with the three cofactors, resulted in a further increase in sedimentation rate, due presumably to an increase in the tetramer-to-dimer ratio. The addition of valine to the complete system resulted in maintenance of the enzyme in the dimeric state concomitant with inhibition of enzyme activity.