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1.
Mol Biochem Parasitol ; 164(1): 45-56, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19073223

ABSTRACT

Immunization with a recombinant yeast-expressed Plasmodium falciparum merozoite surface protein 3 (MSP3) protected Aotus nancymai monkeys against a virulent challenge infection. Unfortunately, the production process for this yeast-expressed material was not optimal for human trials. In an effort to produce a recombinant MSP3 protein in a scaleable manner, we expressed and purified near-full-length MSP3 in Escherichia coli (EcMSP3). Purified EcMSP3 formed non-globular dimers as determined by analytical size-exclusion HPLC with in-line multi-angle light scatter and quasi-elastic light scatter detection and velocity sedimentation (R(h) 7.6+/-0.2nm and 6.9nm, respectively). Evaluation by high-resolution atomic force microscopy revealed non-linear asymmetric structures, with beaded domains and flexible loops that were recognized predominantly as dimers, although monomers and larger multimers were observed. The beaded substructure corresponds to predicted structural domains, which explains the velocity sedimentation results and improves the conceptual model of the protein. Vaccination with EcMSP3 in Freund's adjuvant-induced antibodies that recognized native MSP3 in parasitized erythrocytes by an immunofluorescence assay and gave delayed time to treatment in a group of Aotus monkeys in a virulent challenge infection with the FVO strain of P. falciparum. Three of the seven monkeys vaccinated with EcMSP3 had low peak parasitemias. EcMSP3, which likely mimics the native MSP3 structure located on the merozoite surface, is a viable candidate for inclusion in a multi-component malaria vaccine.


Subject(s)
Antigens, Protozoan/chemistry , Escherichia coli/genetics , Models, Molecular , Plasmodium falciparum/chemistry , Protozoan Proteins/chemistry , Recombinant Proteins/chemistry , Amino Acid Sequence , Animals , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Aotidae , Cloning, Molecular , Gene Expression , Malaria Vaccines/immunology , Malaria, Falciparum/immunology , Malaria, Falciparum/prevention & control , Molecular Sequence Data , Plasmodium falciparum/genetics , Protein Structure, Tertiary , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Random Allocation , Recombinant Proteins/genetics , Vaccines, Synthetic/immunology
2.
Chem Biol ; 14(7): 764-74, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17656313

ABSTRACT

Methionine aminopeptidases (MetAPs) remove the N-terminal initiator methionine during protein synthesis, a prerequisite step for N-terminal myristoylation. N-myristoylation of proto-oncogene c-Src is essential for its membrane association and proper signal transduction. We used bengamides, a family of general MetAP inhibitors, to understand the downstream physiological functions of MetAPs. c-Src from bengamide A-treated cells retained its N-terminal methionine and suffered a decrease in N-terminal myristoylation, which was accompanied by a shift of its subcellular distribution from the plasma membrane to the cytosol. Furthermore, bengamide A decreased the tyrosine kinase activities of c-Src both in vitro and in vivo and eventually delayed cell-cycle progression through G(2)/M. Thus, c-Src is a physiologically relevant substrate for MetAPs whose dysfunction is likely to account for the cell-cycle effects of MetAP inhibitors including bengamide A.


Subject(s)
Aminopeptidases/antagonists & inhibitors , Azepines/pharmacology , Protease Inhibitors/pharmacology , src-Family Kinases/metabolism , Animals , Cell Line , Humans , Methionyl Aminopeptidases , Proto-Oncogene Mas , Proto-Oncogene Proteins pp60(c-src)/metabolism , Subcellular Fractions/enzymology , Subcellular Fractions/metabolism
3.
Biotechnol Lett ; 28(13): 951-8, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16786267

ABSTRACT

An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300-400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.


Subject(s)
Cell Culture Techniques/instrumentation , Chromatography, Ion Exchange/instrumentation , Malaria Vaccines/isolation & purification , Malaria Vaccines/metabolism , Pichia/physiology , Protein Engineering/methods , Protozoan Proteins/isolation & purification , Protozoan Proteins/metabolism , Chromatography, Ion Exchange/methods , Equipment Design , Equipment Failure Analysis , Malaria Vaccines/genetics , Protozoan Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
4.
J Biotechnol ; 121(4): 458-70, 2006 Feb 24.
Article in English | MEDLINE | ID: mdl-16274825

ABSTRACT

Production of recombinant malaria proteins in the methylotrophic yeast Pichia pastoris has been difficult due to constraints in transcription, translation and/or post-translation controls. Use of codon-optimized genes has resolved many of the transcriptional controls; however, efforts to overcome translational and post-translational modifications involving disulfide bond formation and glycosylation have been mostly restricted to knocking-out putative N-linked glycosylation sites. We report now on the effect of overproduction of P. pastoris protein disulfide isomerase (PpPDI) and Plasmodium falciparum (PfPDI) on production of a disulfide-rich P. falciparum transmission-blocking vaccine candidate, Pfs25. Pfs25 is expressed in P. pastoris as two isoforms (A and B); the A form has been selected for Phase I human studies. Overproduction of PpPDI in the P. pastoris Pfs25 production clone markedly enhanced the expression level of Pfs25(A) and (B) by 3-fold, while overproduction of PfPDI increased the proportion of Pfs25(A) compared to (B). The resultant Pfs25 products were purified and fully characterized biochemically. In addition to differences in production levels, the mass spectra of PpPDI-Pfs25(A) compared to Pfs25(A) and PfPDI-Pfs25(A) were different due to the pattern and level of O-linked glycosylation. The overproduction of PpPDI or PfPDI provides new platforms for expression of disulfide-rich malaria proteins.


Subject(s)
Malaria Vaccines/biosynthesis , Pichia/genetics , Plasmodium falciparum/genetics , Protein Disulfide-Isomerases/genetics , Protozoan Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Animals , Gene Expression , Glycosylation , Malaria Vaccines/genetics , Pichia/enzymology , Plasmodium falciparum/enzymology , Protein Folding , Protein Modification, Translational/genetics , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
5.
FEBS Lett ; 576(1-2): 245-50, 2004 Oct 08.
Article in English | MEDLINE | ID: mdl-15474045

ABSTRACT

We have investigated the physiological function of type 2 methionine aminopeptidases (MetAP2) using Caenorhabditis elegans as a model system. A homolog of human MetAP2 was found in the C. elegans genome, which we termed MAP-2. MAP-2 protein displayed methionine aminopeptidase activity and was sensitive to inhibition by fumagillin. Downregulation of map-2 expression by RNAi led to sterility, resulting from a defect in germ cell proliferation. These observations suggest that MAP-2 is essential for germ cell development in C. elegans and that this ubiquitous enzyme may play important roles in a tissue specific manner.


Subject(s)
Aminopeptidases/metabolism , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Cell Division , Germ Cells/physiology , Metalloendopeptidases/metabolism , Amino Acid Sequence , Aminopeptidases/chemistry , Aminopeptidases/drug effects , Aminopeptidases/genetics , Animals , Binding Sites , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/drug effects , Caenorhabditis elegans Proteins/genetics , Conserved Sequence , Cyclohexanes , Down-Regulation , Fatty Acids, Unsaturated/pharmacology , Gene Expression Regulation , Larva , Metalloendopeptidases/chemistry , Metalloendopeptidases/drug effects , Metalloendopeptidases/genetics , Molecular Sequence Data , RNA Interference , Sensitivity and Specificity , Sequence Homology, Amino Acid , Sesquiterpenes
6.
Dev Biol ; 269(2): 319-30, 2004 May 15.
Article in English | MEDLINE | ID: mdl-15110703

ABSTRACT

We are interested in the mechanisms that underlie cell fate determination in the endosporic male gametophytes of the fern, Marsilea vestita. Synchronous development is initiated by placing dry spores into water and involves the translation of stored mRNAs, with little transcription. Nine division cycles produce 32 spermatids surrounded by 7 sterile cells, and then each spermatid differentiates into a multiciliate gamete. Here, we focus on changes in the distribution of particular proteins, mRNAs, and patterns of polyadenylation as essential prerequisites for cell fate determination and gametogenesis. Earlier, we showed that alpha- and beta-tubulin proteins become concentrated in spermatogenous initials, and that centrin mRNA is translated only in spermatogenous initials. In situ hybridizations reveal that centrin, cyclin B, and beta-tubulin mRNAs are present in both sterile and spermatogenous cells, but that transcripts encoding RNA helicase and PRP-19 (a spliceosome component) become localized in spermatogenous cells. The targeted destruction of these two transcripts by RNAi treatments does not affect the numbers of division cycles, but the gametophytes exhibit anomalous patterns of cytokinesis, and a subsequent failure of spermatid differentiation. Thus, cell fate determination in the gametophyte involves localized translation, and the localization of mRNAs for proteins involved in transcript processing. We found differences in polyadenylation levels in sterile and spermatogenous cells that match the distribution of cytoplasmic poly(A) polymerase (PAP), which, in immunolocalizations, is abundant in spermatogenous cells, but undetectable in sterile cells. The activation of translation in spermatogenous initials, but not in sterile cells, may be under the control of mRNA processing enzymes, which become localized either as proteins or mRNAs in the spermatogenous subdomains before any divisions occur.


Subject(s)
Cell Differentiation , Marsileaceae/physiology , RNA, Messenger/analysis , Marsileaceae/cytology , Marsileaceae/genetics , Plant Proteins/analysis , Polyadenylation , RNA, Messenger/metabolism
7.
J Med Chem ; 46(16): 3452-4, 2003 Jul 31.
Article in English | MEDLINE | ID: mdl-12877582

ABSTRACT

Fumagillin and ovalicin constitute a family of structurally related natural products that possess antiangiogenic activity. We report the synthesis of a new fumagillin analogue, fumagalone, in which the spiroepoxide group is replaced with an aldehyde. Fumagalone inhibits type 2 methionine aminopeptidase (MetAP2) with IC(50) = 8 microM and endothelial cell proliferation with IC(50) = 52 nM. With dialysis and competition assays, it was unambiguously demonstrated that binding of fumagalone to MetAP2 is reversible.


Subject(s)
Aminopeptidases/antagonists & inhibitors , Angiogenesis Inhibitors/chemical synthesis , Cyclohexanones/chemical synthesis , Epoxy Compounds/chemical synthesis , Fatty Acids, Unsaturated/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Animals , Cattle , Cell Division/drug effects , Cyclohexanes , Cyclohexanones/chemistry , Cyclohexanones/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Epoxy Compounds/chemistry , Epoxy Compounds/pharmacology , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/pharmacology , In Vitro Techniques , Protein Binding , Sesquiterpenes
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