Protein Engineering Protocols - Mycobacteriology research center

106 Koide and Koideplate. Incubate at 30°C. Observe the color. Pick the clones that show blue color forfurther characterization.3.3.3. Specificity Test of Isolated Monobodies1. Grow yeast cells of interest from library screening in 1.5 mL YPD media at 30°Cfor 16 h and prepare DNA from the yeast cell using Y-DER yeast DNA extractionkit (Pierce).2. Electroporate KC8 E. coli cells with the yeast DNA after dialysis (see Subheading3.1.4.) and select transformants on a LB-Ap plate.3. Replicate the colonies on a minimal trp-Km plate, and incubate at 37°C. Grow twocolonies in 2 mL LB-Ap each at 37°C for up to 10 h and extract plasmid DNAusing standard methods. Using KC8 cells grown more than 10 h often yieldsimpure plasmids. Determine the sequence of the monobody segment by standardDNA-sequencing methods (see Note 7).4. Transform EGY48 with the B42-monobody plasmid, and test its interaction profileagainst various baits using the interaction mating method (Subheading 3.3.1.).3.3.4. Liquid β-Galactosidase AssayBefore biochemical measurements, the affinity between a bait and a monobodycan be characterized semiquantitatively by a liquid-phase β-galactosidase assay.This method is more quantitative than the plate assay described in Subheading3.3.2. We have adapted this method to a 96-well plate format (see Note 8).1. Transform a RFY206 yeast strain with pSH18-34, a LexA-bait plasmid (a derivativeof pEG202), and a B42-monobody plasmid (a derivative of pYesTrp2).Inoculate 2 mL YC Glc his– ura– trp– with a colony and shake overnight at 30°C.2. Remove the media by brief centrifugation and suspend cells in warm YC Gal Rafhis– ura– trp– until the OD 600nmis 0.2. Shake at 30°C for 6 h. Determine theOD 600nmand aliquot at 350 µL each in a microcentrifuge tube. We typically measurein triplicates.3. Mix 2X β-galactosidase assay solution with Y-Per (Pierce) at the ratio of 1 to 1 tomake the working solution. Add 350 µL of the working solution into each sample.Invert several times to mix.4. Incubate at 30°C while checking the color. When a yellow color has developed, add300 µL of 1 N Na 2CO 3into the tubes and mix well. Centrifuge the microcentrifugetubes at the maximal speed for 1 min. Determine the OD 420nmof the supernatant.3.4. Cloning, Expression, Purification, and Biotinylation of a MonobodyAfter identifying a monobody with desired properties from library selection,its gene is transferred to an E. coli expression vector and the monobody isexpressed and purified as an isolated protein. We typically obtain 10 to 50 mgmonobody per liter of culture. We also describe protocols for biotinylating amonobody for easy detection in immunochemical applications.