TABLE OF CONTENTS INTRODUCTION
This set of experiments uses the expression plasmid produced in Part 1 to generate the ?-gal- myo-3 fusion protein. Lab 7.1 begins with a computer simulation exercise that introduces parameters to be considered when setting out to purify a protein of interest. The program simulates some typical decision-making pathways encountered when a researcher attempts to use any of a range of different chromatography procedures, applied in any chosen order by the student.
These lessons are brought to life in the following set of linked experimental exercises. Three linked pathways all begin with Lab 7.2, induction of ?-gal myo-3 fusion protein synthesis using the ?-galactosidase substrate to mimic isopropyl- ?-d-thiogalactoside (IPTG). Next, one of three methods is used to attempt the purification of our expressed myo-3- ?-gal fusion protein, gel filtration chromatography (GFC, Lab 7.3), ion exchange chromatography (IEC, Lab 7.5), and substrate affinity chromatography (AC, Lab 7.6). The goal is to separate the fusion protein from impurities while simultaneously preserving its catalytic activity. The chromatography procedures resolve the input material into a set of separated "fractions" that are each tested ("assayed") to determine if they contain ?-gal- myo-3 fusion protein or not. The presence of protein activity is accomplished on a semi-quantitative basis using the "dot blot" assay in Lab 7.4.
