The reactive triazine dyes: Their usefulness and limitations in protein purifications

Antibodies, interferons, blood clotting factors and enzymes ranging from dehydrogenases and kinases to tRNA synthetases and restriction endonucleases are now purified by chromatography on the immobilized triazine dyes. The range of interactions between the dyes and proteins is so wide that the technique can no longer be termed a truly group-specific affinity chromatographic method. Nevertheless, because the dyeligands are cheap, easy to immobilize and have large capacities for proteins, the method is useful in both preparative and large-scale purifications as an alternative to both conventional and affinity chromatographic techniques.     

Mannosylerythritol lipids: production,downstream processing,and potential applications

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Microchannel enzyme reactors and their applications for processing

Microreaction technology is an interdisciplinary field combining science and engineering. It has attracted the attention of researchers from different fields for the past few years, resulting in the development of several microreactors. Enzymes are one of the catalysts used in microreactors: they are useful for substance production in an environmentally friendly way and have high potential for analytical applications. However, few enzymatic processes have been commercialized because of problems with stability and the cost and efficiency of the reactions. Thus, there have been demands for innovation in process engineering, particularly for enzymatic reactions, and microreaction devices can serve as efficient tools for the development of enzyme processes. In this review, we summarize the recent advances of enzyme-immobilized microchannel reactors; fundamental techniques for micro enzyme-reactor design and important applications of this multidisciplinary technology in chemical processing are also included in our topics.     

Optimal synthesis of chromatographic trains for downstream protein processing

Downstream bioprocessing and especially chromatographic steps, commonly used for the purification of multicomponent systems, are significant cost drivers in the production of therapeutic proteins. There has been an increased interest in the development of systematic methods for the design of such processes, and the appropriate selection of a series of chromatographic steps is still a major challenge to be addressed. Several models have been developed previously but have assumed that 100% recovery of the desired product is obtained at each chromatographic step. In this work, a mathematical framework is proposed, based on mixed integer optimisation techniques, that removes this assumption and allows full flexibility on the position of retention time cut-points, between which the desired product fraction is collected. The proposed model is demonstrated on three example protein mixtures, each containing up to 13 contaminants and selecting from a set of up to 21 candidate steps. The proposed model results in a reduction of one to three chromatographic steps over solutions that no losses are allowed.     

Cyclosporin A--a review on fermentative production, downstream processing and pharmacological applications

In present times, the immunosuppressants have gained considerable importance in the world market. Cyclosporin A (CyA) is a cyclic undecapeptide with a variety of biological activities including immunosuppressive, anti-inflammatory, antifungal and antiparasitic properties. CyA is produced by various types of fermentation techniques using Tolypocladium inflatum. In the present review, we discuss the biosynthetic pathway, fermentative production, downstream processing and pharmacological activities of CyA.     

The metabolism of beta-amyloid converting enzyme and beta-amyloid precursor protein processing

Herein we investigated the processing of beta-secretase (BACE), implicated in Alzheimer's disease through processing of beta-amyloid precursor protein (betaAPP), into smaller metabolites. Four products of approximately 34, approximately 12, approximately 8, and approximately 5kDa were identified, none of which were generated autocatalytically. The approximately 34 and approximately 12kDa forms are held together by disulfide bridges. The approximately 34kDa form results from two cleavages: an N-terminal processing at RLPR(45) downward arrow by furin/PC5, and a C-terminal cleavage at SQDD(379) downward arrow by an unknown enzyme that also releases the C-terminal approximately 12kDa product. Microsequencing of the approximately 8 and approximately 5kDa fragments showed that they are the result of processing at VVFD(407) downward arrow and DMED(442) downward arrow, respectively. Mutagenesis of the identified cleavage sites revealed that the mutants D379A, D379L or D379E block the degradation of BACE into the approximately 12kDa product, confirming the importance of Asp(379). Notably, the D379E mutant results in higher betaAPP derived C99 levels. In contrast, D442A or D442E did not affect the production of the approximately 8 or approximately 5kDa products. The levels of the approximately 8 and approximately 5kDa products are significantly lower in the mutant D407A but less so D407E, likely due to the low efficacy of ER exit of the D407A mutant. Indeed, while co-expression of betaAPP with BACE results in enhanced production of Abeta(11-40), the D407A mutant produces mostly Abeta(40).     

Enzyme stability in downstream processing. Part 1: enzyme inactivation, stability and stabilization

In biotechnological recovery processes the instability of the product can lead to large losses in the sequence of recovery processes needed to purify the product. As the cost of the final active product is strongly dependent on the recovery yield, this will lead to an increase in product cost. Therefore knowledge of factors that influence stability is important. This Part 1 contains a review on the factors that influence stability. As stability is very important in enzyme purification this review deals about enzymes and their ability to retain catalytic activity. Inactivation mechanisms and agents are discussed. A short review is given of enzyme structure and stability. This is followed by stabilization strategies and methods.     

Using chromatography in downstream processing

This article concludes the series on the use of chromatography for downstream processing. Although it has only scratched the surface when considering the number of parameters involved in process chromatography, it does give a broad overview including the choice of components through process standards. Pharmacia LKB Biotechnology has had more than 15 years experience in the design development and running of large scale chromatographic processes. During this time the company has gathered a vast amount of experience and information on the key points to successful product purification. Pharmacia LKB can advise on the choice of techniques and the development of a separation process up to full production scale.     

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.     

The toxicity of textile reactive azo dyes after hydrolysis and decolourisation

The toxicity of C.I. Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the bioluminescent bacterium Vibrio fischeri. Hydrolysed Reactive Black had a slightly greater toxicity than the parent form (EC(50) 11.4+/-3.68 and 27.5+/-4.01 mg l(-1), respectively). A baffled bioreactor with anaerobic and aerobic compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent. Decolourisation of hydrolysed Reactive Black resulted in an increased toxicity (EC(50) 0.2+/-0.03 mg l(-1)). Toxicity was not detectable when decolourised Reactive Black 5 was metabolised under aerobic conditions. No genotoxicity was detected after the decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor. The toxicity and genotoxicity of decolourised C.I. Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC(50) 0.1+/-0.03 mg l(-1)).     

槐糖脂的生物合成、发酵及分离纯化

槐糖脂是一类由酵母菌代谢生产的糖脂型生物表面活性剂,具有优越的表/界面活性和稳定性,且无毒、无刺激、易被生物降解,在众多领域具有良好的应用前景,被认为是最具潜力替代化学表面活性剂的生物表面活性剂之一.历经50余年的研究,槐糖脂的发酵生产工艺日趋成熟,但产品的分离纯化仍是难点.本文系统地综述了槐糖脂的结构、生物合成途径,...     

The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein

The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.     

Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing

The influence of aeration and glucose feeding on the stability of recombinant protein A in Escherichia coli during the transition period from a fed‐batch cultivation to downstream processing was studied. Neither interruption of the feeding under aerobic conditions nor anaerobic conditions in presence of glucose could stabilize protein A completely and the intracellular ATP pool did not decrease to less than 0.75–1 mM by this treatment. On the other hand, the absence of both oxygen and glucose resulted in a decrease of the ATP pool to less than 0.5 mM and almost complete stabilization of protein A. The decrease of ATP was more severe when sulfite was used instead of nitrogen gas to create anaerobic conditions in presence of glucose. This also resulted in nearly complete stabilization of protein A, which might be explained by an inhibiting effect of sodium sulfite on fermentation. Therefore, protein stabilization and decrease of the ATP pool were correlated in experiments in vivo. The concentrations of ADP and AMP increased during starvation and may also play a role in stabilization of the protein in vivo. ATP may be a limiting factor of proteolysis also during further steps of downstream processing. Its concentration decreases by 80–90% during harvesting and centrifugation of biomass and even further during disruption of cells. However, neither addition nor regeneration of ATP in cell disintegrate was enough to restore degradation of protein A, indicating that an additional factor limits proteolysis in vitro. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 730–738, 1999.     

Advances in the production and downstream processing of antibodies

Sales of monoclonal antibody (mAbs) therapies exceeded $ 40 billion in 2010 and are expected to reach $ 70 billion by 2015. The majority of the approved antibodies are targeting cancer and autoimmune diseases with the top 5 grossing antibodies populating these two areas. In addition over 100 monoclonal antibodies are in Phase II and III of clinical development and numerous others are in various pre-clinical and safety studies. Commercial production of monoclonal antibodies is one of the few biotechnology manufacturing areas that has undergone significant improvements and standardization over the last ten years. Platform technologies have been established based on the structural similarities of these molecules and the regulatory requirements. These improvements include better cell lines, advent of high-performing media free of animal-derived components, and advances in bioreactor and purification processes. In this chapter we will examine the progress made in antibody production as well as discuss the future of manufacturing for these molecules, including the emergence of single use technologies.     

The use of dyes in protein purification.

The role of the matrix, ligand and linking mechanism in affinity chromatography is discussed, special emphasis being placed on the use of dyestuff molecules as ligands. Current knowledge of dye-protein interactions is outlined and problems arising from the use of conventional textile dyes as ligands are considered. Work on the synthesis of novel dye-like molecules designed specifically for affinity chromatography is reviewed. This is seen as leading to the development of improved affinity systems capable of advancing the utility of affinity chromatography in protein purification.     

PepN,the major Suc-LLVY-AMC-hydrolyzing enzyme in Escherichia coli,displays functional similarity with downstream processing enzymes in Archaea and eukarya. Implications in cytosolic protein degradation

Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), a fluorogenic endopeptidase substrate, is used to detect 20 S proteasomal activity from Archaea to mammals. An o-phenanthroline-sensitive Suc-LLVY-AMC hydrolyzing activity was detected in Escherichia coli although it lacks 20 S proteasomes. We identified PepN, previously characterized as the sole alanine aminopeptidase in E. coli, to be responsible for the hydrolysis of Suc-LLVY-AMC. PepN is an aminoendopeptidase. First, extracts from an ethyl methanesulfonate-derived PepN mutant, 9218, did not cleave Suc-LLVY-AMC and L-Ala-para-nitroanilide (pNA). Second, biochemically purified PepN cleaves a wide variety of both aminopeptidase and endopeptidase substrates, and L-Ala-pNA is cleaved more efficiently than other substrates. Studies with bestatin, an aminopeptidase-specific inhibitor, suggest differences in the mechanisms of cleavage of aminopeptidase and endopeptidase substrates. Third, PepN hydrolyzes whole proteins, casein and albumin. Finally, an E. coli strain with a targeted deletion in PepN also lacks the ability to cleave Suc-LLVY-AMC and L-Ala-pNA, and expression of wild type PepN in this mutant rescues both activities. In addition, we identified a low molecular weight Suc-LLVY-AMC-cleaving peptidase in Mycobacterium smegmatis, a eubacteria harboring 20 S proteasomes, to be an aminopeptidase homologous to E. coli PepN, by mass spectrometry analysis. "Sequence-based homologues" of PepN include well characterized aminopeptidases, e.g. Tricorn interacting factors F2 and F3 in Archaea and puromycin-sensitive aminopeptidase in mammals. However, our results suggest that eubacterial PepN and its homologues displaying aminoendopeptidase activities may be "functionally similar" to enzymes important in downstream processing of proteins in the cytosol: Tricorn-F1-F2-F3 complex in Archaea and TPPII/Multicorn in eukaryotes.     

The neuropeptide processing enzyme EC 3.4.24.15 is modulated by protein kinase A phosphorylation

The metalloendopeptidase EC (EP24.15) is a neuropeptide-metabolizing enzyme expressed predominantly in brain, pituitary, and testis, and is implicated in several physiological processes and diseases. Multiple putative phosphorylation sites in the primary sequence led us to investigate whether phosphorylation effects the specificity and/or the kinetics of substrate cleavage. Only protein kinase A (PKA) treatment resulted in serine phosphorylation with a stoichiometry of 1.11 +/- 0.12 mol of phosphate/mol of recombinant rat EP24.15. Mutation analysis of each putative PKA site, in vitro phosphorylation, and phosphopeptide mapping indicated serine 644 as the phosphorylation site. Phosphorylation effects on catalytic activity were assessed using physiological (GnRH, GnRH(1-9), bradykinin, and neurotensin) and fluorimetric (MCA-PLGPDL-Dnp and orthoaminobenzoyl-GGFLRRV-Dnp-edn) substrates. The most dramatic change upon PKA phosphorylation was a substrate-specific, 7-fold increase in both K(m) and k(cat) for GnRH. In both rat PC12 and mouse AtT-20 cells, EP24.15 was serine-phosphorylated, and EP24.15 phosphate incorporation was enhanced by forskolin treatment, and attenuated by H89, consistent with PKA-mediated phosphorylation. Cloning of the full-length mouse EP24.15 cDNA revealed 96.7% amino acid identity to the rat sequence, and conservation at serine 644, consistent with its putative functional role. Therefore, PKA phosphorylation is suggested to play a regulatory role in EP24.15 enzyme activity.