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Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

ABSTRACT: Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe.

SUBMITTER: Steere AN

PROVIDER: S-EPMC2860049 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

Steere Ashley N AN Roberts Samantha E SE Byrne Shaina L SL Dennis Chasteen N N Bobst Cedric E CE Kaltashov Igor A IA Smith Valerie C VC MacGillivray Ross T A RT Mason Anne B AB

Protein expression and purification 20100111 1

Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombi ...[more]

PMID: 20064616

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Project description:The liver possesses an ability of compensatory growth after removing three of five lobes in mammals or one of three lobes in zebrafish. The reenter of hepatocytes into the cell cycle is one of the hallmarks for the initiation of liver compensatory growth, but cellular and molecular mechanisms underlying the activation of hepatocytes remain largely unknown.To better understand the process, transcriptional profiles of the remaining liver dorsal lobes in female zebrafish were generated with RNA-seq. About 44 million raw reads were obtained from three sequencing libraries and 71 % of raw reads were mapped to the reference genome of zebrafish. A total number of 5652 genes were differentially expressed in at least one of two time points during the compensatory growth of liver dorsal lobes and classified into different functional categories. A number of genes encoding angiogenesis-related growth factors/ligands and apoptosis-associated cytokines were strongly expressed at 6-h time point after the removal of the ventral lobe. Gene ontology enrichment analysis of genes up-regulated during early stages of liver compensatory growth revealed that small GTPase-mediated signal transduction, RNA processing and intracellular protein transport were the most highly overrepresented biological processes and SNARE interactions in vesicular transport, proteasome and basal transcription factors were the most highly enriched pathways. Moreover, 477 genes differently expressed during liver compensatory growth of both female zebrafish and mice were involved in the response to stimulus, DNA replication, metabolic processes of fatty acid, lipid and steroid, multicellular organismal homeostasis and extracellular matrix constituent secretion.Multiple biological processes and signaling pathways are immediately activated in remaining dorsal lobes of female zebrafish right after removal of the ventral lobe and these findings provide crucial clues for further identification of cis-elements and trans-factors that are extensively involved in the initiation of liver compensatory growth.

Dataset Information

Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

Publications

Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

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