Publications (10)20.14 Total impact
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Chapter: BACE Biological Assays
03/2010: pages 35 - 58; , ISBN: 9780470594087 -
Article: The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus.
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ABSTRACT: Compounds capable of stimulating soluble guanylate cyclase (sGC) activity might become important new tools to treat hypertension. While rational design of these drugs would be aided by elucidation of the sGC three-dimensional structure and molecular mechanism of activation, such efforts also require quantities of high quality enzyme that are challenging to produce. We implemented the titerless infected-cells preservation and scale-up (TIPS) methodology to express the heterodimeric sGC. In the TIPS method, small-scale insect cell cultures were first incubated with a recombinant baculovirus which replicated in the cells. The baculovirus-infected insect cells (BIIC) were harvested and frozen prior to cell lysis and the subsequent escape of the newly replicated virus into the culture supernatant. Thawed BIIC stocks were ultimately used for subsequent scale up. As little as 1 mL of BIIC was needed to infect a 100-L insect cell culture, in contrast to the usual 1L of high-titer, virus stock supernatants. The TIPS method eliminates the need and protracted time for titering virus supernatants, and provides stable, concentrated storage of recombinant baculovirus in the form of infected cells. The latter is particularly advantageous for virus stocks which are unstable, such as those for sGC, and provides a highly efficient alternative for baculovirus storage and expression. The TIPS process enabled efficient scale up to 100-L batches, each producing about 200mg of active sGC. Careful adjustment of expression culture conditions over the course of several 100-L runs provided uniform starting titers, specific activity, and composition of contaminating proteins that facilitated development of a process that reproducibly yielded highly active, purified sGC.Protein Expression and Purification 02/2009; 65(2):122-32. · 1.59 Impact Factor -
Article: Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells.
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ABSTRACT: Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships.Protein Expression and Purification 02/2009; 65(2):133-9. · 1.59 Impact Factor -
Article: Potent and selective isophthalamide S2 hydroxyethylamine inhibitors of BACE1.
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ABSTRACT: The design and synthesis of a novel series of potent BACE1 hydroxyethylamine inhibitors. These inhibitors feature hydrogen bonding substituents at the C-5 position of the isophthalamide ring with improved selectivity over cathepsin D.Bioorganic & Medicinal Chemistry Letters 07/2007; 17(12):3378-83. · 2.55 Impact Factor -
Article: Design of potent inhibitors of human beta-secretase. Part 2.
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ABSTRACT: We describe an optimized series of acyclic hydroxyethylamine transition state isosteres of beta-secretase that incorporates a variety of P(2) side chains that yield potent inhibitors with excellent cellular activity. A 2.2A crystal structure of compound 13 is shown.Bioorganic & Medicinal Chemistry Letters 02/2007; 17(1):78-81. · 2.55 Impact Factor -
Article: Design of potent inhibitors of human beta-secretase. Part 1.
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ABSTRACT: We describe a novel series of potent inhibitors of human beta-secretase. These compounds possess the hydroxyethyl amine transition state isostere. A 2.5A crystal structure of inhibitor 32 bound to BACE is provided.Bioorganic & Medicinal Chemistry Letters 02/2007; 17(1):73-7. · 2.55 Impact Factor -
Article: Human beta-secretase (BACE) and BACE inhibitors.
Journal of Medicinal Chemistry 11/2003; 46(22):4625-30. · 5.25 Impact Factor -
Article: Human β-Secretase (BACE) and BACE Inhibitors
09/2003; -
Article: Employing a superior BACE1 cleavage sequence to probe cellular APP processing.
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ABSTRACT: The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.Journal of Neurochemistry 04/2003; 84(5):1006-17. · 4.06 Impact Factor -
Article: Cloning, sequencing and phylogenetic analysis of a human 5-hydroxytryptamine1D receptor pseudogene
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ABSTRACT: A third member of the human 5HTID gene family has been identified using a combination of homology cloning and DNA sequence analysis. This human gene is most related to the 5HT1Dα subtype (77% shared identity) and is a pseudogene, based on the lack of an open reading frame (ORF) caused by multiple in-frame stop codons and nucleotide (nt) deletions relative to the functional 5HT1Dα gene (encoding the 5-hydroxytryptamine1Dα receptor). The 5HT1D pseudogene also contained an insertion that shares 87% identity to the Alu consensus sequence. Phylogenetic analysis of the three human genes in this family reveals that although the two functional genes, 5HT1Dα and 5HT1Dgb, are detected in all mammalian species examined, the 5HT1D pseudogene is only detected in a subset of primates (catarrhines) that evolved approximately 35–45 million years (Myr) ago. Alternatively, based on the 23% divergence between the functional 5HT1Dα gene and the 5HT1D pseudogene, we estimate that these two genes began to diverge approximately 50 Myr ago.Gene.
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2010
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Buck Institute for Research on Aging
Novato, CA, USA
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