Beta-galactosidase

Some interesting facts Muller-Hill-1996


 * 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 &beta;-galactosidase. Beckwith-1970
 * 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 &beta;-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 &beta;-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 Plovins-ApplEnvironMicrobio-1994
 * Another fluorogenic substrate is 4-methylumbelliferyl &beta;-D-galactopyranoside (MUG) which works in bacteria, yeast, and mammalian cells (without requiring permeabilization/lysis). Vidal-Aroca-2006
 * Alpha-complementation of &beta;-galactosidase does not seem to yield activities equal to wildtype &beta;-galactosidase. Depending on the fragment, the activity can be up to 24% of wildtype Zamenhof-JBacteriol-1972 . (If anyone has a better reference comparing results from a Miller assay of alpha-complementated &beta;-galactosidase with wildtype, please include it here.)

Protocols

 * beta-galactosidase assay
 * LacZ staining of cells

Reference
// original &beta;-galactosidase assay by Miller
 * 1) Muller-Hill-1996 isbn=3-11-014830-7
 * 2) Beckwith-1970 isbn=0317118099
 * 3) Ullmann-Bioessays-1992 pmid=1345751
 * 4) Plovins-ApplEnvironMicrobio-1994 pmid=7811104
 * 5) Miller-1972 isbn=0879691069
 * 1) Zamenhof-JBacteriol-1972 pmid=4552986
 * 2) Thibodeau-Biotechniques-2004 pmid=15038156
 * 3) WorthingtonBiochem http://www.worthington-biochem.com/BG/default.html
 * 4) Serebriiskii-AnalBiochem-2000 pmid=10998258
 * 5) Vidal-Aroca-2006 pmid=16629389