GSM8825396: MMP1 Low affinity sub-population rep 3; Saccharomyces cerevisiae; OTHER
The sorted N-TIMP2 library fractions (high and low affinity) were isolated from the yeast cells using the E.Z.N. A. Yeast Plasmid Mini Kit (Omega Bio-tek, Norcross, GA, U.S.A.) according to the manufacturer's protocol. The library, purchased from GenScript (Piscataway, NJ), was constructed using NNS degenerate codons (where N represents A, C, T, or G nucleotides, and S represents C or G) at positions 4, 35, 38, 68, 71, 97, and 99 on the basis of the N-TIMP2WT gene (PDB ID code 1BUV), giving random mutationsat a single position for each clone. This single position N-TIMP2 mutagenesis library was expressed in a YSD system with the Saccharomyces cerevisiae EBY100 yeast strain according to an established protocol. For the construction of a yeast-displayed N-TIMP2 library with a free N-terminus, the pCHA-VRC01-scFv vector  was used. In this construct, the C-terminus of the N-TIMP2 library was fused to the N-terminus of Aga-2, leaving the N-terminus of the displayed N-TIMP2 protein variants exposed to the solvent. The pCHA vector was linearized with the restriction enzymes BamHI-HF and Nhe-HF and, together with the N-TIMP2 library, were transformed by homologous recombination into a competent EBY100 S. cerevisiae yeast strain using a MicroPulser electroporator.. A library size of 5.45 × 10^6 transformants was obtained, as determined by plating serial dilutions on SDCAA plates (2% dextrose, 0.67% yeast nitrogen base, 0.5% Bacto casamino acids, 1.54% Na2HPO4, 1.856% Na2HPO4·H2O, 18.2% sorbitol, and 1.5% agar).