Beta-galactosidase

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

• beta-galactosidase assay
• LacZ staining of cells

See also

• X-Gal

Reference

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1. ISBN:3-11-014830-7
2. ISBN:0317118099
7. ISBN:0879691069

   original β-galactosidase assay by Miller

8. Thibodeau SA, Fang R, and Joung JK. High-throughput beta-galactosidase assay for bacterial cell-based reporter systems. Biotechniques. 2004 Mar;36(3):410-5. DOI:10.2144/04363BM07 |

9. http://www.worthington-biochem.com/BG/default.html