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Replicase
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
The first hfq gene mutants were constructed and characterized by a chromosomal insertion mutation (Tsui et al. 1994). The hfq mutation exhibited a wide range of growth defects and phenotypes that resembled those reported for bacteria containing mutant histone-like proteins. These results confirmed the critical physiological role of the Hfq in E. coli. A functional complementation of the hfq gene product to the E. coli hns gene product was found especially interesting because of the frequent copurification of the hns gene product during the Hfq purification process (Shi and Bennett 1994).
Intracellular Peptide Turnover: Properties and Physiological Significance of the Major Peptide Hydrolases of Brain Cytosol
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
The identity of the two enzymes was confirmed in a series of studies by Barrett and Tisljar who found that both activities were identical to Pz-peptidase, an enzyme known for many years. Pz-peptidase is an enzyme that is widely distributed in mammalian tissues. It owes its name to its action on the synthetic peptide substrate phenylazobenzyloxycarbonyl (Pz)-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide), designed as a substrate to detect collagenase-like activity. On the basis of biochemical and immunological criteria, Tisljar et al. demonstrated that Pz-peptidase is identical to endo-oligopeptidase A111. On the basis of co-purification, substrate specificity, and the action of inhibitors, Barrett and Tisljar concluded that Pz-peptidase and the soluble metalloendopeptidase were identical as well112. The name “thimet oligopeptidase” now replaces all of the earlier names for this enzyme15.
Structure, Function, and Regulation of Pulmonary Surfactant Proteins
Published in Jacques R. Bourbon, Pulmonary Surfactant: Biochemical, Functional, Regulatory, and Clinical Concepts, 2019
Jeffrey A. Whitsett, Timothy E. Weaver
The close association of SP-A with surfactant phospholipids is an inherent property noted during its co-purification with surfactant phospholipids from lung lavage. SP-A binds to phospholipids and enhances their aggregation in the presence of calcium.98 Calcium alters the conformation of SP-A as well; the intrinsic fluorescence of tryptophan residues located in the carboxy terminus of SP-A are altered by the addition of calcium.99 Changes in calcium-dependent phospholipid aggregation and intrinsic tryptophan fluorescence occur at calcium concentrations of 0.2 to 0.5 mM, consistent with the concentrations of calcium in the extracellular space in the alveolus. Recent studies have demonstrated that the generation of tubular myelin forms by mixtures containing SP-A, SP-B, and phospholipids require the presence of calcium.84
Technical advancement and practical considerations of LC-MS/MS-based methods for host cell protein identification and quantitation to support process development
Published in mAbs, 2023
Jia Guo, Regina Kufer, Delia Li, Stefanie Wohlrab, Midori Greenwood-Goodwin, Feng Yang
Trace-level HCPs can lead to undesirable product instability, immune responses, or formulation changes, and they need to be removed to a sufficient level. Process development to mitigate these risks requires sensitive methods to understand the HCP population and the interaction between HCP and antibody.30,31 LC-MS/MS-based proteomics approaches to profile HCPs from harvested cell culture fluid (HCCF) to the final drug substance allow us to understand how abundant or problematic HCPs are removed across different process steps. Generally, HCP patterns in HCCF are relatively similar across CHO cell lines, while HCP patterns during purification and product concentration become more divergent; therefore, MS can be valuable to detect individual HCPs in downstream processes and guide purification development efforts.17,32 For example, protein identification and quantitation by size exclusion chromatography (SEC) fractionation coupled to LC-MS/MS showed HCPs (like high-risk hydrolytic enzymes) co-eluting with high molecular weight (HMW) species – this indicated the co-purification of HCPs with mAb and that reducing HMW species in drug substance might significantly decrease the levels of some HCPs.33
Current challenges in biotherapeutic particles manufacturing
Published in Expert Opinion on Biological Therapy, 2020
Mafalda G. Moleirinho, Ricardo J.S. Silva, Paula M. Alves, Manuel J. T. Carrondo, Cristina Peixoto
Regarding large scale production, continuous flow ultracentrifugation is also becoming a well-established approach to concentrate and purify the virus in one step, as it allows for processing of large volumes [46–48]. Nevertheless, these ultracentrifugation techniques are poorly scalable, difficult to maintain, due to the size and price of the equipment, and allow for the co-purification of some impurities [49]. Recently, a new CsCl density gradient method using general centrifugation (40,000 x g) was described, instead of the conventional method (100,000 x g), for virus purification [50]. The use of a common centrifuge instead of ultracentrifuges reduces the cost and becomes a more accessible virus purification method. Besides the disadvantages of ultracentrifugation, this strategy is still used to produce small quantities of the highly purified virus at lab scale or even for phase I clinical trials. However, in the past few years, the industry is trying to move away from these centrifugation processes and focus on chromatography and membrane-based separation techniques.
SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level
Published in mAbs, 2020
Christel Aebischer-Gumy, Pierre Moretti, Romain Ollier, Christelle Ries Fecourt, François Rousseau, Martin Bertschinger
In the simplest cases, the protein of interest can be directly tagged, for example, with green fluorescent protein (GFP).5 However, protein function and structure may be altered by the presence of the tag. Therefore, direct tagging is usually not applicable for therapeutic proteins. Other methods rely on the co-expression of a fluorescent protein (e.g., GFP) with the gene of interest, either through an internal ribosome entry site (IRES), using polycistronic vectors or by co-transfection.6–14 However, limitations in the secretory pathway, protein folding or different efficiencies in translation may affect the correlation of expression of the reporter protein and the gene of interest. These limitations are partially overcome by using a transmembrane reporter protein co-expressed with the protein of interest, such as using an IRES in the expression cassette,14–18 or due to a non-optimal start codon,19 even if an expression bias, which could be caused by uncorrelated expression of reporter and gene of interest due to different folding kinetics, may still be possible. In general, the reporter protein needs to be carefully selected in order to avoid toxic effects for the host cell20 or co-purification with the gene of interest.