Dataset Information

Development of a reporter peptide that catalytically produces a fluorescent signal through ?-complementation.

ABSTRACT: In ?-complementation, inactive N-terminal (?-domain) and C-terminal (?-domain) fragments of ?-galactosidase associate to reconstitute the active protein. To date, the effect of ?-domain size on ?-complementation activity has not been systematically investigated. In this study, we compared the complementation activities of ?-domains of various sizes using an in vitro system. We found that the complementation activities are similar for ?-domains comprising between 45 and 229 N-terminal residues but are significantly decreased for those containing less than 37 residues. However, these smaller ?-domains (15 and 25 residues) exhibited sufficient ?-complementation activity for application as reporters.

SUBMITTER: Nishiyama K

PROVIDER: S-EPMC4420511 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a reporter peptide that catalytically produces a fluorescent signal through α-complementation.

Nishiyama Kotaro K Ichihashi Norikazu N Kazuta Yasuaki Y Yomo Tetsuya T

Protein science : a publication of the Protein Society 20150402 5

In α-complementation, inactive N-terminal (α-domain) and C-terminal (ω-domain) fragments of β-galactosidase associate to reconstitute the active protein. To date, the effect of α-domain size on α-complementation activity has not been systematically investigated. In this study, we compared the complementation activities of α-domains of various sizes using an in vitro system. We found that the complementation activities are similar for α-domains comprising between 45 and 229 N-terminal residues bu ...[more]

PMID: 25740628

Similar Datasets

Project description:Formyl-methionine-containing peptides (e.g. fMet-Leu-Phe) stimulate a variety of neutrophil functions by interacting with specific cell surface receptors which are coupled via G-proteins to stimulation of phospholipase C. Two markedly distinct cDNAs coding for formyl peptide receptors have recently been isolated from a rabbit and a human cDNA library respectively. To examine the hitherto unknown signal transduction properties of the formyl peptide receptor encoded by the human cDNA, we have used the PCR to clone this cDNA from poly(A)+ RNA of myeloid differentiated human leukaemia (HL-60) cells, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes. Receptor activity was determined electrophysiologically by measuring the agonist-dependent opening of intracellular Ca2+ concentration ([Ca2+]i)-independent Cl- channels. Injection of pure formyl peptide receptor cRNA did not lead to peptide-dependent changes in membrane current. In contrast, marked alterations of membrane current were observed in response to formyl peptides when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells. Injection of the latter RNA did not lead to formyl-peptide-dependent alterations of membrane current. Binding studies using a radioiodinated formyl peptide revealed that injection of formyl peptide receptor cRNA alone led to expression of the formyl peptide receptor on the oocyte surface, and that co-injection of poly(A)+ RNA from undifferentiated HL-60 cells did not alter the level of receptor expression. Size fractionation of poly(A)+ RNA from undifferentiated HL-60 cells showed that the mRNA required to complement formyl-peptide-dependent signal transduction in oocytes had a size of approx. 3-3.5 kb. These results strongly suggest that the human formyl peptide receptor requires a specific cofactor(s), which is lacking in Xenopus oocytes but is present in undifferentiated HL-60 cells, to activate the second messenger pathway in oocytes. Identification of this factor will provide important information about the molecular mechanisms by which G-protein-coupled granulocyte-activating receptors stimulate phospholipase C.

Dataset Information

Development of a reporter peptide that catalytically produces a fluorescent signal through ?-complementation.

Publications

Development of a reporter peptide that catalytically produces a fluorescent signal through α-complementation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets