ABSTRACT
Session 1 of the 2019 Viral Clearance Symposium proposed strategies to use prior knowledge for demonstrating robust viral clearance for biotechnologically produced recombinant proteins, such as monoclonal antibodies, and introduced methods for virus reduction applicable to recombinant adeno-associated virus (rAAV) vectors. Proposals for generic virus clearance claims were made for detergent inactivation with Triton CG-110, low pH inactivation, anion-exchange membrane chromatography, and virus filtration, and acceptance of these proposals were discussed. Furthermore, the development of an ASTM standard for retrovirus removal by small virus filters was presented, and it was found that updating and refining of conditions is still warranted. The current approach for log reduction value calculation by using the lower value from duplicate runs as worst case was questioned, and a modified concept was proposed. Finally, for rAAV vectors, several options for reduction of adventitious viruses were demonstrated such as 35 nm virus filtration, detergent inactivation, heat inactivation, and anion-exchange or affinity chromatography and were found to be feasible in order to satisfy the principles of ICH Q5A.
Subject(s)
Drug Contamination , Viruses , Chromatography , Detergents , Drug Contamination/prevention & control , FiltrationABSTRACT
Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.
Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Cytosol/chemistry , Endoplasmic Reticulum/metabolism , Microsomes/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Saccharomyces cerevisiae/metabolismABSTRACT
Chemical modification of ion channels has recently attracted attention due to their potential use in stochastic sensing and neurobiology. Among the available channel templates stable ß-barrel proteins have shown their potential for large scale chemical modifications due to their wide pore lumen. Ion-channel hybrids using the outer membrane protein OmpG were generated by S-alkylation with a synthetic modulator and functionally as well as structurally characterized. The dansyl moiety of the used modulator resulted in partial blockage of current though the OmpG channel with its gating characteristics mainly unaffected. The crystal structure of an OmpG-dansyl hybrid at 2.4Å resolution correlates this finding by showing that the modulator lines the inner walling of the OmpG pore. These results underline the suitability of OmpG as a structural base for the construction of stochastic sensors.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Escherichia coli Proteins/chemistry , Porins/chemistry , Alkylation , Amino Acid Substitution , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Crystallography, X-Ray , Dansyl Compounds/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutation , Porins/genetics , Porins/metabolism , Protein Structure, TertiaryABSTRACT
The attachment of modulators to a trimeric porin ion channel was investigated (see picture of the trimer with a crown ether modulator (orange)). The interplay of modulator and protein is essential for the conformational heterogeneity of the hybrid channel. Single-site attachment in large pores is not sufficient to change the electrophysiological characteristics of the pores-such change requires additional noncovalent interactions or second-site attachments.
Subject(s)
Porins/chemistry , Crown Ethers/chemistry , Crystallography, X-Ray , Ion Channels/chemistry , Protein ConformationABSTRACT
2-(Hydroxymethyl)glutarate dehydrogenase, the fourth enzyme of the anaerobic nicotinate fermentation pathway of Eubacterium barkeri, catalyzes the NADH-dependent conversion between (S)-2-formylglutarate and (S)-2-(hydroxymethyl)glutarate. As shown by its 2.3-A crystal structure, this enzyme is a novel member of the beta-hydroxyacid dehydrogenase family and adopts a tetrameric architecture with monomers interacting via their C-terminal catalytic domains. The NAD-binding domains protrude heterogeneously from the central, tetrameric core with domain rotation angles differing up to 12 degrees. Kinetic properties of the enzyme, including NADH inhibition constants, were determined. A strong NADH binding in contrast to weaker NAD(+) binding of the protein was inferred from fluorometrically determined binding constants for the dinucleotide cofactor. The data support either an Iso Ordered Bi Bi mechanism or a more common Ordered Bi Bi mechanism as found in other dehydrogenases.