Generation of IL-23 Alanine-scanning mutagenesis using yeast display technology

Correct assembly of IL-23 heterodimer requires co-expression and co-localization of p19 and p40 subunits on the surface of Saccoromyces cerevisiae yeast cells. To accomplish this, we cloned the p40 gene into a soluble-expression plasmid (proprietary pYKY vector) containing a uracil selection marker under the control of a constitutive promotor. The p19 subunit was cloned into a surface-display plasmid (proprietary pTS6 vector) with a tryptophan selection marker under the control of a galactose-inducible promoter. The p19 subunit is expressed as an Aga1 fusion protein with a modified GPI anchor with V5 tag, which was used to confirm protein expression. The display vector was designed to allow alanine mutants of the p19 subunit to be constructed using Kunkel mutagenesis. Specific reverse compliment oligonucleotides (from IDT) encoding alanine at each position were annealed to a single-strand uracil-containing phagemid vector with wild-type p19. Complementary strand was synthesized using T4 DNA polymerase (NEB) and the resulting duplex was treated with T4 DNA ligase (Roche). The constructs were transformed into competent E. coli cells (Invitrogen), grown under selection conditions, and confirmed for successful integration by sequence analysis. The constructs were transformed into naïve yeast cultures using lithium acetate/ssDNA/PEG method. BJ5464 cells (ATCC) are grown on YPD (Teknova) plates for 2–3 days following an initial thaw and plating. A small patch of cells is used to inoculate 10 ml of YPD broth (Teknova) in a vented conical tube (Corning) and these cells are grown at 30°C at 200 rpm in an incubator shaker overnight. Cell density was measured with a spectrophotometer and diluted to OD₆₀₀ of 0.2 in YPD (in a volume determined for the number of transformations needed) in vented Erlenmeyer flasks (Corning) and grown at 30°C for ~4 hrs until the OD₆₀₀ of 0.8–1.2 is reached. The cells are centrifuged gently, washed once with water, and resuspended at approximately 1e8 cells in 50 μl in individual Eppendorf tubes. Following the removal of the water by brief centrifugation, the following components are added to cells in the following order: 240 μl PEG3500 (at 50%w/v; Sigma), 36 μl 1 M lithium acetate (Sigma), 50 μl ssDNA (1 mg/ml; denatured), 36 μl of plasmid DNA plus d/d water. The optimal amounts of p19 display vector and p40 secretion vector were determined to be 800 ng and 200 ng, respectively. The cells with the transformation mix are vortexed vigorously for one minute then incubated at 42°C for one hour with an occasional gentle mix. The transfection mix is removed following centrifugation, and the cells are resuspended in water and plated out onto CM/glucose plates without uracil or tryptophan (Teknova). The plates are sealed and grown for 3 to 4 days in a 30°C incubator, thereafter stored at 4°C. Individual colonies are picked into 10 ml of CM glucose Trp^-/Ura^- broth (Teknova) in vented conical tubes and grown overnight at 30°C at 200 rpm in an incubator shaker. The following day approximately 3 ml of this growth stage culture is collected and following media removal, resuspended in 10 ml CM galactose Trp^-/Ura^- media (Teknova). These cultures are grown at 20°C at 200 rpm in an incubator shaker overnight. Typically 100 μl of the induced cultures are added to each well of a deep-well block (Thomson) for staining of membrane associated proteins and specific antigens.