12th International Symposium on Bioluminescence & Chemiluminescence

Symposium abstracts:

Random mutagenesis of firefly luciferase to identify amino acid residues affecting substrate binding

Southworth, T.L., Murtiashaw, M.H., Ruggiero, M.C., Branchini, B.R.

Dept. of Chemistry, Connecticut College, New London, CT 06320 USA

Email: tlsou@conncoll.edu

The functions of specific amino acids can be investigated by site directed mutagenesis. Random mutagenesis techniques, such as error prone PCR, introduce mutations at random sites. Stratagene’s GeneMorph PCR mutagenesis kit was used to amplify a product containing the luciferase gene. An error prone DNA polymerase introduced mutations at low, moderate, and high frequencies, creating mutational libraries of luciferase. The libraries were screened by in vivo assays using luciferin or luciferin analogs, including 6´-aminoluciferin and quinolylluciferin, to identify clones with significantly altered bioluminescent activity. Preliminary results indicated that libraries of low and high mutational frequency exhibited decreased bioluminescent intensity. DNA sequencing of clones with modified bioluminescence will determine the amino acid changes introduced and indicate amino acid residues that warrant further study.

This is a preprint of an article accepted for publication in Luminescence: Copyright 2001 John
Wiley & Sons, Ltd (Wiley website)