-
PloS One 2013Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein...
Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.
Topics: Alkaline Phosphatase; Biological Therapy; Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Proteins; Immunochemistry; Mass Spectrometry
PubMed: 24312330
DOI: 10.1371/journal.pone.0081639 -
The Journal of Cell Biology Apr 2000The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that...
The Nup84p complex consists of five nucleoporins (Nup84p, Nup85p, Nup120p, Nup145p-C, and Seh1p) and Sec13p, a bona fide subunit of the COPII coat complex. We show that a pool of green fluorescent protein-tagged Sec13p localizes to the nuclear pores in vivo, and identify sec13 mutant alleles that are synthetically lethal with nup85Delta and affect the localization of a green fluorescent protein-Nup49p reporter protein. In the electron microscope, sec13 mutants exhibit structural defects in nuclear pore complex (NPC) and nuclear envelope organization. For the assembly of the complex, Nup85p, Nup120p, and Nup145p-C are essential. A highly purified Nup84p complex was isolated from yeast under native conditions and its molecular mass was determined to be 375 kD by quantitative scanning transmission electron microscopy and analytical ultracentrifugation, consistent with a monomeric complex. Furthermore, the Nup84p complex exhibits a Y-shaped, triskelion-like morphology 25 nm in diameter in the transmission electron microscope. Thus, the Nup84p complex constitutes a paradigm of an NPC structural module with distinct composition, structure, and a role in nuclear mRNA export and NPC bio- genesis.
Topics: Alleles; Amino Acid Sequence; Cell Nucleus; Chromatography, Gel; Epistasis, Genetic; Fungal Proteins; Genes, Lethal; Membrane Proteins; Microscopy, Electron, Scanning Transmission; Molecular Sequence Data; Molecular Weight; Mutation; Nuclear Envelope; Nuclear Pore Complex Proteins; Nuclear Proteins; Porins; Protein Binding; RNA-Binding Proteins; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Temperature; Ultracentrifugation
PubMed: 10747086
DOI: 10.1083/jcb.149.1.41 -
Methods in Enzymology 2017Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid...
Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid phosphatidylethanolamine. Atg8 lipidation is mediated by distinctive UBL cascades involving autophagy-specific E1, E2, and E3 enzymes that differ substantially in sequence from components of other UBL conjugation cascades. Structural studies are important for elucidating the roles of Atg proteins that regulate multiple steps involved in autophagy. This chapter describes methods to prepare and crystallize selected proteins and complexes involved in autophagy UBL conjugation pathways, as a guide for strategies for structural and biochemical characterization of Atg proteins.
Topics: Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Autophagy-Related Proteins; Crystallization; Crystallography; Humans; Mutation; Protein Domains; Saccharomyces cerevisiae Proteins; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases
PubMed: 28253958
DOI: 10.1016/bs.mie.2016.09.058 -
Journal of Molecular Biology Jul 2008The yeast Rap1 protein plays an important role in transcriptional silencing and in telomere length homeostasis. Rap1 mediates silencing at the HM loci and at telomeres...
The yeast Rap1 protein plays an important role in transcriptional silencing and in telomere length homeostasis. Rap1 mediates silencing at the HM loci and at telomeres by recruiting the Sir3 and Sir4 proteins to chromatin via a Rap1 C-terminal domain, which also recruits the telomere length regulators, Rif1 and Rif2. We report the 1.85 A resolution crystal structure of the Rap1 C-terminus, which adopts an all-helical fold with no structural homologues. The structure was used to engineer surface mutations in Rap1, and the effects of these mutations on silencing and telomere length regulation were assayed in vivo. Our surprising finding was that there is no overlap between mutations affecting mating-type and telomeric silencing, suggesting that Rap1 plays distinct roles in silencing at the silent mating-type loci and telomeres. We also found novel Rap1 phenotypes and new separation-of-function mutants, which provide new tools for studying Rap1 function. Yeast two-hybrid studies were used to determine how specific mutations affect recruitment of Sir3, Rif1, and Rif2. A comparison of the yeast two-hybrid and functional data reveals patterns of protein interactions that correlate with each Rap1 phenotype. We find that Sir3 interactions are important for telomeric silencing, but not mating type silencing, and that Rif1 and Rif2 interactions are important in different subsets of telomeric length mutants. Our results show that the role of Rap1 in silencing differs between the HM loci and the telomeres and offer insight into the interplay between HM silencing, telomeric silencing, and telomere length regulation. These findings suggest a model in which competition and multiple recruitment events modulate silencing and telomere length regulation.
Topics: Amino Acid Sequence; Carrier Proteins; Chromosome Mapping; Chromosomes, Fungal; Crystallography, X-Ray; Models, Biological; Models, Molecular; Molecular Sequence Data; Mutation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Shelterin Complex; Silent Information Regulator Proteins, Saccharomyces cerevisiae; Telomere; Telomere-Binding Proteins; Transcription Factors; Two-Hybrid System Techniques
PubMed: 18538788
DOI: 10.1016/j.jmb.2008.04.078 -
Nature Communications Aug 2019Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone...
Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.
Topics: Acetylation; Catalytic Domain; Cell Cycle Proteins; Histone Acetyltransferases; Histone Chaperones; Histones; Intrinsically Disordered Proteins; Lysine; Molecular Chaperones; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Protein Processing, Post-Translational; Recombinant Proteins; Saccharomyces cerevisiae Proteins; Substrate Specificity; Xenopus Proteins
PubMed: 31387991
DOI: 10.1038/s41467-019-11410-7 -
Molecular & Cellular Proteomics : MCP May 2013Malaria infections of mammals are initiated by the transmission of Plasmodium salivary gland sporozoites during an Anopheles mosquito vector bite. Sporozoites make their...
Malaria infections of mammals are initiated by the transmission of Plasmodium salivary gland sporozoites during an Anopheles mosquito vector bite. Sporozoites make their way through the skin and eventually to the liver, where they infect hepatocytes. Blocking this initial stage of infection is a promising malaria vaccine strategy. Therefore, comprehensively elucidating the protein composition of sporozoites will be invaluable in identifying novel targets for blocking infection. Previous efforts to identify the proteins expressed in Plasmodium mosquito stages were hampered by the technical difficulty of separating the parasite from its vector; without effective purifications, the large majority of proteins identified were of vector origin. Here we describe the proteomic profiling of highly purified salivary gland sporozoites from two Plasmodium species: human-infective Plasmodium falciparum and rodent-infective Plasmodium yoelii. The combination of improved sample purification and high mass accuracy mass spectrometry has facilitated the most complete proteome coverage to date for a pre-erythrocytic stage of the parasite. A total of 1991 P. falciparum sporozoite proteins and 1876 P. yoelii sporozoite proteins were identified, with >86% identified with high sequence coverage. The proteomic data were used to confirm the presence of components of three features critical for sporozoite infection of the mammalian host: the sporozoite motility and invasion apparatus (glideosome), sporozoite signaling pathways, and the contents of the apical secretory organelles. Furthermore, chemical labeling and identification of proteins on live sporozoites revealed previously uncharacterized complexity of the putative sporozoite surface-exposed proteome. Taken together, the data constitute the most comprehensive analysis to date of the protein expression of salivary gland sporozoites and reveal novel potential surface-exposed proteins that might be valuable targets for antibody blockage of infection.
Topics: Animals; Anopheles; Calcium Signaling; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Female; Host-Parasite Interactions; Insect Vectors; Membrane Proteins; Mice; Plasmodium falciparum; Plasmodium yoelii; Proteome; Proteomics; Protozoan Proteins; Salivary Glands; Secretory Pathway; Sporozoites
PubMed: 23325771
DOI: 10.1074/mcp.M112.024505 -
Journal of Proteome Research Oct 2012Human nonintegrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. The laminin...
Human nonintegrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. The laminin receptor is overexpressed in several human cancers and is also the cell surface receptor for several viruses and pathogenic prion proteins, making it a pathologically significant protein. This study focused on the proteomic characterization of laminin receptor interacting proteins from Mus musculus. The use of affinity chromatography with immobilized recombinant laminin receptor coupled with mass spectrometry analysis identified 45 proteins with high confidence. Following validation through coimmunoprecipitation, the proteins were classified based on predicted function into ribosomal, RNA processing, signal transduction/metabolism, protein processing, cytoskeleton/cell anchorage, DNA/chromatin, and unknown functions. A significant portion of the identified proteins is related to functions or localizations previously described for laminin receptor. This work represents a comprehensive proteomic approach to studying laminin receptor and provides an essential stepping stone to a better mechanistic understanding of this protein's diverse functions.
Topics: Animals; Chaperonin Containing TCP-1; Chromatography, Affinity; Cytoskeletal Proteins; Hexosyltransferases; Histones; Humans; Intracellular Signaling Peptides and Proteins; Mice; NIH 3T3 Cells; Proteasome Endopeptidase Complex; Protein Binding; Proteome; Proteomics; RNA-Binding Proteins; Receptors, Laminin; Ribosomal Proteins; Transcription Factors
PubMed: 22909348
DOI: 10.1021/pr300307h -
Nucleic Acids Research May 2012Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross...
Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5-Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35-54) and putative Prp40-binding (PPxY(100)) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382-476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273-286 and 299-312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146-269) are essential pairwise (Ile198-Arg190; Leu256-Leu259) or in trios (Leu169-Arg172-Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions between the Mud2-Msl5 complex at the branchpoint and the U1 snRNP at the 5' splice site.
Topics: Amino Acid Motifs; Amino Acid Sequence; Guanosine; Models, Molecular; Molecular Sequence Data; Mutation; Protein Structure, Tertiary; RNA Splicing Factors; RNA-Binding Proteins; Ribonucleoprotein, U1 Small Nuclear; Ribonucleoproteins; Ribonucleoproteins, Small Nuclear; Saccharomyces cerevisiae Proteins; Splicing Factor U2AF; Structure-Activity Relationship
PubMed: 22287628
DOI: 10.1093/nar/gks049 -
The Journal of Biological Chemistry May 1998H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the...
H-kininogen is a multifunctional protein: it inhibits cysteine proteases, plays a role in contact activation of the coagulation cascade, and is the precursor of the potent proinflammatory peptide bradykinin. In the experiments described here, we identify H-kininogen as a ferritin-binding protein. Ferritin is a cellular and serum protein that is elevated in acute and chronic inflammation and many cancers. Despite numerous reports of ferritin-binding protein(s) in human serum, the nature and function of these proteins remain unclear. As a first step in characterizing the interaction between ferritin and its binding protein(s), we devised a ligand blot assay and used it to guide purification of a ferritin-binding protein from human serum. Edman degradation of the purified protein determined the sequence HNLGHGHK(H)ERDQGHG, a sequence with identity to residues 421-436 of human H-kininogen. These results were confirmed by demonstrating that commercially purified H-kininogen possessed ferritin binding activity and that ferritin binding could not be detected in plasma from kininogen-deficient individuals. Ligand blot assays mapped the ferritin binding domain to the light chain of H-kininogen chain, and revealed that both H and L recombinant ferritins possess H-kininogen binding activity. The unexpected identification of H-kininogen as a ferritin-binding protein may link ferritin in the complex chain of interactions by which H-kininogen mediates its multiple effects in contact activation and inflammation.
Topics: Amino Acid Sequence; Blotting, Western; Ferritins; Humans; Iron-Binding Proteins; Kininogens; Ligands; Molecular Sequence Data; Protein Binding; Receptors, Cell Surface
PubMed: 9593701
DOI: 10.1074/jbc.273.22.13630 -
The Biochemical Journal Sep 2003Pyruvate occupies a central role in energy homoeostasis, and dysregulation of its cellular disposition underlies many metabolic disturbances. Although the mitochondrial... (Review)
Review
Pyruvate occupies a central role in energy homoeostasis, and dysregulation of its cellular disposition underlies many metabolic disturbances. Although the mitochondrial membrane pyruvate transporter has been characterized, its molecular identity has proved elusive. Recent work has now identified a single candidate protein for the mitochondrial pyruvate carrier in yeast, opening the way for further studies in mammalian systems, which may have important therapeutic applications within the context of metabolic disease.
Topics: Animals; Anion Transport Proteins; Humans; Membrane Transport Proteins; Mitochondrial Membrane Transport Proteins; Mitochondrial Proteins; Monocarboxylic Acid Transporters; Pyruvic Acid; Saccharomyces cerevisiae Proteins
PubMed: 12954079
DOI: 10.1042/bj20031105