Beta-galactosidase

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Some interesting facts [1]

  • A tetramer of 4 identical subunits
  • Each subunit is 120kD.
  • Active only as a tetramer.
  • Mutations in some of the codons of the N-terminal 60aa or C-terminal 100aa results in an inactive, dimeric β-galactosidase. [2]
  • If the above sequences are deleted, the missing protein fragment can be replaced by the corresponding peptide. This is called intracistronic alpha or omega complementation respectively.
  • Its N-terminal 23 residues can be replaced by any amino acid residues without affecting the enzymatic activity.
  • A mutant with an internal deletion of codons 21-41 of the lacZ gene does not produce any active β-galactosidase.
  • A mutant with a deletion of everything past residue 60 (i.e. it expresses only the first 60 N-terminal amino acids) does not produce any active β-galactosidase.
  • You can measure lacZ activity using flow cytometry. See A flow cytometric study of stationary phase gene expression in E.coli using lacZ reporter gene fusion
    • This paper uses C12FDG from Invitrogen. However, FDG might be a better substrate in gram-negative bacteria like Escherichia coli [3]
  • Another fluorogenic substrate is 4-methylumbelliferyl β-D-galactopyranoside (MUG) which works in bacteria, yeast, and mammalian cells (without requiring permeabilization/lysis). [4]
  • Alpha-complementation of β-galactosidase does not seem to yield activities equal to wildtype β-galactosidase. Depending on the fragment, the activity can be up to 24% of wildtype [5]. (If anyone has a better reference comparing results from a Miller assay of alpha-complementated β-galactosidase with wildtype, please include it here.)

Protocols

See also

Reference

  1. isbn:3-11-014830-7. [Muller-Hill-1996]
  2. isbn:0317118099. [Beckwith-1970]
  3. Plovins A, Alvarez AM, Ibañez M, Molina M, and Nombela C. . pmid:7811104. PubMed HubMed [Plovins-ApplEnvironMicrobio-1994]
  4. Vidal-Aroca F, Giannattasio M, Brunelli E, Vezzoli A, Plevani P, Muzi-Falconi M, and Bertoni G. . pmid:16629389. PubMed HubMed [Vidal-Aroca-2006]
  5. Zamenhof PJ and Villarejo M. . pmid:4552986. PubMed HubMed [Zamenhof-JBacteriol-1972]
  6. Ullmann A. . pmid:1345751. PubMed HubMed [Ullmann-Bioessays-1992]
  7. [by] Jeffrey H. Miller. Experiments in molecular genetics. [Cold Spring Harbor, N.Y.] Cold Spring Harbor Laboratory, 1972. isbn:0879691069. [Miller-1972]
    original β-galactosidase assay by Miller

  8. Thibodeau SA, Fang R, and Joung JK. . pmid:15038156. PubMed HubMed [Thibodeau-Biotechniques-2004]
  9. http://www.worthington-biochem.com/BG/default.html

    [WorthingtonBiochem]

  10. Serebriiskii IG and Golemis EA. . pmid:10998258. PubMed HubMed [Serebriiskii-AnalBiochem-2000]
All Medline abstracts: PubMed HubMed
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