Amino acid sequence of beta-galactosidase. IV. Sequence of an alpha-complementing cyanogen bromide peptide, residues 3 to 92.

Langley, KE; Fowler, A V; Zabin, Irving

doi:10.1016/s0021-9258(19)41641-4

Cited by 53 publications

(6 citation statements)

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“…The α-complementation of β-galactosidase is a well-known reaction in which two parts of the enzyme, an inactive N-terminal deletion mutant of the enzyme [enzyme acceptor (EA)] and the inactive deleted peptide [enzyme donor (ED)], associate to generate an active enzymatic species. − This construct is commonly utilized both in the blue-white screening technique for bacteria and in CEDIA immunoassays . This reaction is also utilized in the detection of in vitro and in vivo protein–protein interactions and in high-throughput screening assays, , among other applications. , In this paper, we examine the α-complementation reaction of β-galactosidase at the single-molecule level to characterize the active species that are generated in the complementation reaction and compare their activity to that of the native β-galactosidase enzyme.…”

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confidence: 99%

Stoichiometry of the α-Complementation Reaction of Escherichia coli β-Galactosidase As Revealed through Single-Molecule Studies

Mogalisetti

Walt

2015

Biochemistry

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The α-complementation reaction of β-galactosidase was studied at single-molecule resolution using arrays of femtoliter-sized wells. Single molecules of the complementation species were observed to be stable for long periods of time, demonstrating that the α-complementation reaction is irreversible. By directly counting the number of active molecules formed in the complementation reaction when different concentrations of enzyme acceptor (EA) and enzyme donor (ED) are used, we deduce that the EA:ED ratio in the complementation species is 4:1.

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confidence: 99%

Stoichiometry of the α-Complementation Reaction of Escherichia coli β-Galactosidase As Revealed through Single-Molecule Studies

Mogalisetti

Walt

2015

Biochemistry

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“…CNBr digestion of wild-type β-galactosidase yielded a fragment that also brought about complementation (Lin et al, 1970) of a mutant β-galactosidase. The peptide was made up of residues 3-92 of the wild-type β-galactosidase (Langley et al, 1975a). The complementation of β-galactosidase (called R-complementation) is usually done with M15 β-galactosidase (Langley & Zabin, 1976).…”

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confidence: 99%

Monomer−Dimer Equilibrium of Uncomplemented M15 β-Galactosidase from Escherichia coli

Gallagher

Huber

1997

Biochemistry

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A series of gel filtration, native polyacrylamide gel electrophoresis (PAGE) and sucrose density experiments showed that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium and that only under some specific conditions does the equilibrium strongly favor dimerization. The ratio of dimer to monomer increased as a function of the protein concentration, and a very good fit to a theoretical plot of the effect of protein concentration on an associating system of this type was found. The Kdiss (equilibrium constant for dimer dissociation) was 2.5 x 10(-7) M. The addition of 20 mM Mg2+ lowered the Kdiss to 1.5 x 10(-7) M, and the addition of 150 mM NaCl lowered the value to 0.4 x 10(-7) M. Thiol reagents (2-mercaptoethanol and dithiothreitol) caused the equilibrium to shift totally to the dimeric form. The monomer-dimer equilibrium was also found to be dependent upon the pH. The dissociation increased as the pH was raised to 8.5, but there was a reversal of the equilibrium in favor of dimer formation at pH 9.0. This suggests that one (or more) residues with a pKa value of about 8.0 is involved. Tyr and Lys were eliminated as possible residues involved and it is, therefore, likely that one or more Cys are involved. Further evidence that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium was that the gel-filtration peaks were not totally resolved and that native PAGE bands were diffuse under all conditions except at high thiol concentration.

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“…Individually, the monomer fragments are small, stable, and enzymatically inactive. When the correct combination of β-gal fragments are incorporated in bacteria or correctly oriented in mammalian cells, trans -complementation occurs and enzymatic activity is restored. − Further, we demonstrate the utility of these fragments for targeted-complementation in live cell assays and demonstrate the robustness of the system with an eye on utilizing these fragments to investigate molecular signatures associated with disease.…”

Section: Introductionmentioning

confidence: 81%

“…The two fragments are inactive separately. But when an alpha-donor subunit links two alpha-acceptor dimers together, intracistronic complementation (dimer−dimer interaction) occurs and restores the active quaternary conformation of β-gal. − …”

Section: Introductionmentioning

confidence: 99%

Expanding the Utility of β-Galactosidase Complementation: Piece by Piece

et al. 2009

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The ability to image and quantify multiple biomarkers in disease necessitates the development of split reporter fragment platforms. We have divided the β-galactosidase enzyme into unique, independent polypeptides that are able to re-assemble and complement enzymatic activity in bacteria and in mammalian cells. We created two sets of complementing pairs that individually have no enzymatic activity. However, when brought into close geometric proximity, the complementing pairs associated resulting in detectable enzymatic activity. We then constructed a stable ligand complex comprised of reporter fragment, linker, and targeting moiety. The targeting moiety, in this case a ligand, allowed cell surface receptor targeting in vitro. Further, we were able to simultaneously visualize two cell surface receptors implicated in cancer development, epidermal growth factor receptor and transferrin receptor, using complementing pairs of the ligand-reporter fragment complex.

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Amino acid sequence of beta-galactosidase. IV. Sequence of an alpha-complementing cyanogen bromide peptide, residues 3 to 92.

Cited by 53 publications

References 29 publications

Stoichiometry of the α-Complementation Reaction of Escherichia coli β-Galactosidase As Revealed through Single-Molecule Studies

Stoichiometry of the α-Complementation Reaction of Escherichia coli β-Galactosidase As Revealed through Single-Molecule Studies

Monomer−Dimer Equilibrium of Uncomplemented M15 β-Galactosidase from Escherichia coli

Expanding the Utility of β-Galactosidase Complementation: Piece by Piece

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