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]
• 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 [4]. (If anyone has a better reference comparing results from a Miller assay of alpha-complementated β-galactosidase with wildtype, please include it here.)

Protocols

1. Beta-Galactosidase Assay (A better Miller)
2. BE.109:Protein engineering/Assessing beta-galactosidase

Reference

1. ISBN:3-11-014830-7
2. ISBN:0317118099
3. Plovins A, Alvarez AM, Ibañez M, Molina M, and Nombela C. Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells. Appl Environ Microbiol. 1994 Dec;60(12):4638-41. DOI:10.1128/aem.60.12.4638-4641.1994 |
4. Zamenhof PJ and Villarejo M. Construction and properties of Escherichia coli strains exhibiting -complementation of -galactosidase fragments in vivo. J Bacteriol. 1972 Apr;110(1):171-8. DOI:10.1128/jb.110.1.171-178.1972 |
5. Ullmann A. Complementation in beta-galactosidase: from protein structure to genetic engineering. Bioessays. 1992 Mar;14(3):201-5. DOI:10.1002/bies.950140311 |
6. ISBN:0879691069

   original β-galactosidase assay by Miller

7. 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 |