Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.
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PMID:In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. 136 26

The 1.3 S biotinylatable subunit of Proprionibacterium shermanii transcarboxylase complex was fused to the C-terminus of the human neurokinin 1 receptor gene and introduced into the Semliki Forest virus expression vector pSFV1. RNA transcribed from pSFV1-NK1-biot and pSFV-Helper2 was coelectroporated into BHK cells permitting in vivo packaging of recombinant virus. Infection of BHK and CHO cells with SFV-NK1-biot virus yielded high level of the fusion receptor as detected by metabolic labeling, immunoblotting with streptavidin alkaline phosphatase and binding to substance P. Like native receptor, the biotinylated receptor fusion was able to stimulate Ca2+ mobilization in infected CHO cells, indicating functional coupling to guanine-nucleotide-binding proteins.
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PMID:Functional activity of a biotinylated human neurokinin 1 receptor fusion expressed in the Semliki Forest virus system. 788 38

Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.
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PMID:Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion. 865 79