The influence of high hydrostatic pressure on cocaine and veratrine action in a vertebrate nerve

The application of high hydrostatic pressure to toad sciatic nerve causes a gain in sodium and a loss of potassium which are not affected by cocaine. However, cocaine action is enhanced by high pressure when counteracting veratrine depolarization and when blocking the action potential. Various effects of elevated pressure on the after-potentials are presented and the role of ions in these processes is discussed.     

MINLP models for the synthesis of optimal peptide tags and downstream protein processing

The development of systematic methods for the synthesis of downstream protein processing operations has seen growing interest in recent years, as purification is often the most complex and costly stage in biochemical production plants. The objective of the work presented here is to develop mathematical models based on mixed integer optimization techniques, which integrate the selection of optimal peptide purification tags into an established framework for the synthesis of protein purification processes. Peptide tags are comparatively short sequences of amino acids fused onto the protein product, capable of reducing the required purification steps. The methodology is illustrated through its application on two example protein mixtures involving up to 13 contaminants and a set of 11 candidate chromatographic steps. The results are indicative of the benefits resulting by the appropriate use of peptide tags in purification processes and provide a guideline for both optimal tag design and downstream process synthesis.     

Sarcolectin: complete purification for molecular cloning.

A great variety of vertebrate cells contain detectable amounts of lectins, able to stimulate the initiation of cellular DNA synthesis. One of them, sarcolectin (SCL) can block interferon (IFN) action, by inhibiting the synthesis and the expression of the IFN dependent secondary proteins. As a result, the IFN-induced antiviral state is abolished in the cells, which likely facilitates their replication. We identified a major 65 kDa and a minor 55 kDa protein, which could carry these cellular functions. Their purification, especially that of the 65 kDa, was difficult, because of the proximity of albumin. We devised therefore a two-step primary separation, followed by a four-step final purification, which are reported here. The purification was controlled by high pressure liquid chromatography (HPLC), SDS-PAGE electrophoresis and identified by Western blots. We found that only the minor 55 kDa protein can be considered as being sarcolectin, while the major 65 kDa band results from the binding of some SCL molecules to albumin. The major biological functions, namely, stimulation of DNA synthesis and cell agglutination were preserved to the end of the last purification step. This work is requisite for establishing the molecular structure of SCL by recombinant DNA technology.     

Purification of lipases.

Interest on lipases from different sources (microorganisms, animals and plants) has markedly increased in the last decade due to the potential applications of lipases in industry and in medicine. Microbial and mammalian lipases have been purified to homogeneity, allowing the successful determination of their primary aminoacid sequence and, more recently, of the three-dimensional structure. The X-ray studies of pure lipases will enable the establishment of the structure-function relationships and contribute for a better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters. This article reviews the separation and purification techniques that were used in the recovery of microbial, mammalian and plant lipases. Several purification procedures are analysed taking into account the sequence of the methods and the number of times each method is used. Novel purification methods based on liquid-liquid extraction, membrane processes and immunopurification are also reviewed.     

Synthesis of the 3'-thio-nucleosides and subsequent automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage

Oligodeoxynucleotides containing 3'-S-phosphorothiolate (3'-PS) linkages have become useful tools for probing enzyme-catalyzed cleavage processes in DNA. This protocol describes the synthesis of the phosphorothioamidite monomers derived from thymidine and 2'-deoxycytidine, and their application to a fully automated procedure for synthesising oligodeoxynucleotides containing 3'-PS linkages. The synthesis of the 5'-protected-3'-amidites is achievable in 2 weeks with the DNA synthesis and purification taking another 1 week.     

Pressure-induced conformational changes in an antigen and an antibody and the implications on their use for hyperbaric immunoadsorption.

Pressure-induced conformational changes in two proteins, bovine serum albumin (BSA) and immunoglobulin G (IgG), were studied to assess the application of hyperbaric manipulation to the dissociation of antigen-antibody complexes. Antigen-antibody dissociation is important in the product-recovery phase of immunoadsorption, an affinity purification process. Three techniques were used in parallel for this study, including fluorescence, Fourier transform infrared (FTIR) spectroscopy, and the enzyme-linked immunosorbent assay (ELISA). Employing a fluorescent probe, fluorescent intensity measurements were used to detect protein conformational changes. FTIR spectroscopy was used to determine changes in protein secondary structure induced by high pressure, while the ELISA test was used to examine antibody recognition after the proteins had been pressure-treated. The results from this work demonstrate that IgG is resistant to conformational changes induced by pressures below 2 kbar. In contrast, BSA undergoes reversible conformational changes in this pressure range. However, these conformational changes are not reflected in tests measuring antibody recognition. These findings indicate that IgGs have the potential to be used as recycled ligands in immunoadsorption separation processes. Different antigens that are being considered for purification by immunoadsorption and separated by means of high pressure could be screened by the methods disclosed to determine their stability under high pressure conditions.     

Efficient MILP formulations for the optimal synthesis of chromatographic protein purification processes

The use of recombinant proteins has increased greatly in recent years, as well as the techniques used for their purification. The selection of an efficient process to purify proteins is a major bottleneck found when trying to scale up results obtained in the laboratory to a large-scale industrial process. One of the main challenges in the synthesis of downstream purification stages in biotechnological processes is the appropriate selection and sequencing of chromatographic steps. The objective of this work is to develop mixed integer linear programming models for the synthesis of protein purification processes. Models for each chromatographic technique rely on physicochemical data of a protein mixture, which contains the desired product and provide information on its potential purification. Formulations that are based on convex hull representations are proposed to calculate the minimum number of steps from a set of chromatographic techniques that must achieve a specified purity level and alternatively to maximize purity for a given number of steps. The proposed models are tested in several examples with experimental data and present time reductions of up to three orders of magnitude when compared to big-M formulations.     

Optimal synthesis of protein purification processes

There has been an increasing interest in the development of systematic methods for the synthesis of purification steps for biotechnological products, which are often the most difficult and costly stages in a biochemical process. Chromatographic processes are extensively used in the purification of multicomponent biotechnological systems. One of the main challenges in the synthesis of purification processes is the appropriate selection and sequencing of chromatographic steps that are capable of producing the desired product at an acceptable cost and quality. This paper describes mathematical models and solution strategies based on mixed integer linear programming (MILP) for the synthesis of multistep purification processes. First, an optimization model is proposed that uses physicochemical data on a protein mixture, which contains the desired product, to select a sequence of operations with the minimum number of steps from a set of candidate chromatographic techniques that must achieve a specified purity level. Since several sequences that have the minimum number of steps may satisfy the purity level, it is possible to obtain the one that maximizes final purity. Then, a second model that may use the total number of steps obtained in the first model generates a solution with the maximum purity of the product. Whenever the sequence does not affect the final purity or more generally does not impact the objective function, alternative models that are of smaller size are developed for the optimal selection of steps. The models are tested in several examples, containing up to 13 contaminants and a set of 22 candidate high-resolution steps, generating sequences of six operations, and are compared to the current synthesis approaches.     

New developments in affinity chromatography

The design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.     

Hydrostatic pressure-induced changes in cellular protein synthesis

Hydrostatic pressure is a well-known effector of cellular protein synthesis. High continuous hydrostatic pressure inhibits protein synthesis in general. It has been known for a long time that 30S ribosomal subunit is associated with the effects of pressure on protein synthesis in prokaryotes, however, the mechanisms of action are still not completely understood. Our new data suggest that synthesis of eukaryotic elongation factor-2 (eEF-2) is decreased under 30 MPa continuous hydrostatic pressure. Thus, eEF-2 may have a role in the synthesis of pressure-regulated proteins in eukaryotic cells. The presence of pressure-sensitive proteins indicate that hydrostatic pressure can induce very specific responses in stressed cells. Accumulation of heat shock protein 70 and 90 beta occurs under high pressure, independent of the general inhibition of protein synthesis, although this response appears clearly weaker than during heat stress.     

Polysaccharide-based polymer blends: Methods of their production

The existing methods of preparing polymer blends of cellulose, chitin and chitosan with natural and synthetic polymers and their applications are reviewed. The methods of solid-phase blending of these polymers under conditions of joint action of high pressure and shear deformation are discussed. Normally, under these conditions the processes of dispersion of polymer particles, amorphization, mixing at different levels, depolymerization as well as a chemical interaction resulting in formation of branched and crosslinked structures can take place. The probability and intensity of these processes depend in many respects on the type and magnitude of the external force, but the properties of the polymers are of higher importance. The advantages of the method of joint action of high pressure and shear deformation compared to the conventional techniques of polysaccharides mixtures production are shown.     

Microscale to manufacturing scale-up of cell-free cytokine production--a new approach for shortening protein production development timelines

Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins.     

吡咯喹啉醌合成及生产工艺研究进展

吡咯喹啉醌(pyrroloquinoline quinone, PQQ)是继烟酰胺和核黄素之后发现的第三类氧化还原酶辅因子,普遍存在于生物体中参与呼吸链电子传递,具有促进线粒体产生、清除自由基、增强细胞代谢和预防心肌损伤等生理功能,在医药、食品和农业领域具有广泛的应用前景。微生物发酵法是PQQ生产的主要方式,解析PQQ生物合成途径及其调控机制,通过代谢工程选育短周期、高产量的生产菌是PQQ工业化的研究方向之一。本文综述了PQQ的合成途径、高产菌株选育以及微生物发酵生产与分离纯化的研发工作,为深入阐释PQQ的生物合成机制和工业化生产菌株的选育提供参考。     

Continuous preparative liquid chromatography in the downstrem processing of biotechnological products

Continuous counter‐current chromatographic processes have been successfully used in the petrochemical and sugar industry over the last 30 years. Only recently has simulated moving bed (SMB)‐technology attracted widespread interest in the pharmaceutical industry, mainly as a very efficient system for chromatographic enantioseparation. The application of this technique to the downstream processing of biotechnological products requires some specific changes to meet the special demands of bioproduct isolation. Production processes are set up on an multi‐ton scale, for example, for the purification of fructose with both yield and purity higher than 90%. Examples for other mono‐ and oligosaccharides are reported. In the purification of fatty acids or fat soluble vitamins, SMB technology under supercritical fluid conditions gives additional benefits and increases the productivity by a factor of four when a pressure gradient is applied. Another field of operation is the isolation of drug compounds from natural sources where different batch‐ and SMB‐chromatographic steps could be successfully combined. First examples are reported for cyclosporine A and paclitaxel isolation. Finally, step‐gradient elution modes can be used continuously, as demonstrated for the isolation of monoclonal antibodies.     

Participation of protein synthesis in brain cell structures in the action of antifeins on long-term memory]

Incorporation of 3H-leucine into proteins of rat brain cell structures during application of antifeins (compounds of alternative action on memory processes) has been studied. No correlation was observed between changes in protein synthesis in nuclei, mitochondria, components of endoplasmic reticulum and memory effects of ethyl-, allyl- and propylnorantifeins. Only M1 and M2-demethylated structural analogs of ethylnorantifeins (exerting the most effective action on RNA synthesis and retention of conditional reflexes) enhanced the synaptosomal protein synthesis.     

A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography

Unquestionably, the purification of polypeptides by chromatographic methods is a considerable bottleneck in their preparation. Peptides synthesised by solid phase synthesis typically contain chromatographically similar impurities that complicate purification by reversed phase high performance liquid chromatography (HPLC) techniques. We report on the application of a slow gradient HPLC protocol that allows, in a single chromatographic step, the purification of hundreds of milligrammes of material. This technique was applied to an extensive collection of synthetic polypeptides some incorporating non‐proteinogenic functionality. In all cases examined, the peptides were not only obtained in high purity peptides but were also recovered in multi‐milligramme amounts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with nonstandard synthesis, deprotection, or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and use.     

Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities,and purification of the dye

Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.     

Convergent solid-phase peptide synthesis. VIII. Synthesis, using a photolabile resin, and purification of a methionine-containing protected peptide

The solid-phase synthesis, photolytic detachment from the solid support and purification in solution, of a fully-protected octapeptide containing a methionine residue (protected as the sulphoxide) is described. Protection of methionine in this manner avoids problems associated with the oxidation of this residue during the photolysis. The peptide has been purified by medium pressure liquid chromatography using solvent mixtures containing a high proportion of dimethylformamide in order to avoid precipitation of the peptide on the column.     

F-18 Labeling Protocol of Peptides Based on Chemically Orthogonal Strain-Promoted Cycloaddition under Physiologically Friendly Reaction Conditions

We introduce the high-throughput synthesis of various (18)F-labeled peptide tracers by a straightforward (18)F-labeling protocol based on a chemo-orthogonal strain-promoted alkyne azide cycloaddition (SPAAC) using aza-dibenzocyclootyne-substituted peptides as precursors with (18)F-azide synthon to develop peptide based positron emission tomography (PET) molecular imaging probes. The SPAAC reaction and subsequent chemo-orthogonal purification reaction with azide resin proceeded quickly and selectively under physiologically friendly reaction conditions (i.e., toxic chemical reagents-free, aqueous medium, room temperature, and pH ≈7), and provided four (18)F-labeled tumor targetable bioactive peptides such as cyclic Arg-Gly-Asp (cRGD) peptide, bombesin (BBN), c-Met binding peptide (cMBP), and apoptosis targeting peptide (ApoPep) in high radiochemical yields as direct injectable solutions without any HPLC purification and/or formulation processes. In vitro binding assay and in vivo PET molecular imaging study using the (18)F-labeled cRGD peptide also demonstrated a successful application of our (18)F-labeling protocol.