BE.109:Protein engineering

Module 2

Instructor: Natalie Kuldell

TA: Maria Foley

In this experimental module you will study an enzyme with a remarkable history, beta-galatosidase. "Beta-gal" as it's affectionately called had a starring role in the development of the operon model for gene regulation and continues to be a lab workhorse for gene expression studies. In bacteria, this enzyme hydrolyzes the disaccharide, lactose, into two simpler sugars, glucose and galactose. You will be measuring the efficiency of the enzymatic reaction using an artificial substrate, ONPG, which yields a yellow product when it is cleaved by beta-gal. Using a specialized bacterial strain, you will overexpress beta-gal to purify and analyze. Finally, you will test the effect of replacing a natural amino acid in beta-gal with an unnatural amino acid of your choosing, looking at the effect of such a substitution on the expression and activity of the modified enzyme.

Lab handouts

Day 1: Tools for protein engineering

Day 2: Assessing beta-galactosidase

Day 3: Purifying beta-galactosidase

Day 4: Possible topics for student presentations

References

Note: PDF reprints are provided below within the context of fair use. Please obtain copies from the publisher if appropriate.

1. A new code for life
   EMBO Reports 2004 5(4):336-339
   Rinaldi A.
   URL
2. The structure of E. coli beta-galactosidase
   CR Biol 2005 328(6):549-556
   Matthews B.W.
   URL