Preparation of DNA Inserts and Vector for Library Construction Bio-Rad MyCycler? thermal cycler (Bio-Rad). AccuPrime? Pfx DNA Polymerase (Invitrogen). Plasmid isolation kit (Qiagen). Gel extraction kit (Qiagen). Primers shown in Table 1. SfiI restriction enzyme (New England BioLabs). Yeast display vector pYD7 (see Note 1). 2.3. the yeast display system, the antibody or protein is displayed on the yeast surface by fusing to the yeast agglutinin protein Aga2p, which attaches to Aga1p through two disulfide bonds. Expression of the antibody/protein-Aga2p and Aga1p are under the control of galactose-inducible GAL1 promoter (1). One of the main advantages this technology offers is its eukaryotic system providing very sophisticated protein folding and chaperones machinery, which allows efficient and consistent display of variety of proteins. Recently, yeast surface display has been successfully used to engineer not only antibodies (2C5), but also a wide variety of proteins like fibronectin (6, 7), T cell receptors (TCRs) (8), natural killer cell receptors (9), and proteins of the major histocompatibility complex (MHC) (10) with dramatic improvements in stability and affinity. More importantly, a high-efficacy yeast electroporation protocol has been described (11) recently, which enables construction of yeast display library with large size up to PDK1 inhibitor 1010. This makes yeast display comparable to phage display system in terms of library size and thus further simplifies the initial antibody or protein isolation process. Isolated or engineered human immunoglobulin constant 1 CH2 domains were proposed as novel scaffolds for construction of libraries containing diverse binders of small size but still conferring some effector functions (12C15). Here, we describe a protocol for displaying, engineering, and characterization of CH2 domains against antigens of interest using yeast display (Fig. 1). The yeast-displayed CH2 mutant library with size at 109 was generated by transformation of yeast with linearized vector and mutant DNA inserts derived from CH2 domain through E.coli polyclonal to GST Tag.Posi Tag is a 45 kDa recombinant protein expressed in E.coli. It contains five different Tags as shown in the figure. It is bacterial lysate supplied in reducing SDS-PAGE loading buffer. It is intended for use as a positive control in western blot experiments error-prone PCR. One round of magnetic-activated cell sorting (MACS) against the biotinylated antigen was performed first to downsize the initial library. The library was then sorted several times by fluorescent-activated cell sorting (FACS) to enrich for specific binders. The enriched CH2 domain mutants were further mutated by error-prone PCR of the entire gene pool to yield a new sub-library. The process of sorting and mutagenesis was then cyclically repeated until the cell population reached the desired property. Clones with the highest affinity from the final round of PDK1 inhibitor maturation were identified and their sequences were analyzed. Open in a separate window Fig. 1. Sketch of the protocol for selection and maturation of binders. 2.?Materials 2.1. Error-Prone PCR Bio-Rad MyCycler? thermal cycler (Bio-Rad). GeneMorph? II Random Mutagenesis Kit (Agilent). 8-Oxo-deoxyguanosine triphosphate and 2-deoxy-p-nucleoside-5-triphosphate. Gel extraction kit (Qiagen). Primers shown in Table 1. Table 1 PCR primers used PDK1 inhibitor ERRORF5 CTTCAGTTTTGGCCCAGGCGGCC 3ERRORR5 ACCACTAGTTGGGCCGGCCTG 3YDRDF5 CTTCGCTGTTTTTCAATATTTTCTGTTATTGCTTCAGTTTTGGCCCAGGCGGCC 3YDRDR5 GAGCCGCCACCCTCAGAACCGCCACCCTCAGAGCCACCACTAGTTGGGCCGGCCTG 3 Open in a separate PDK1 inhibitor window 2.2. Preparation of DNA Inserts and Vector for Library Construction Bio-Rad MyCycler? thermal cycler (Bio-Rad). AccuPrime? Pfx DNA Polymerase (Invitrogen). Plasmid isolation kit (Qiagen). Gel extraction kit (Qiagen). Primers shown in Table 1. SfiI restriction enzyme (New England BioLabs). Yeast display vector pYD7 PDK1 inhibitor (see Note 1). 2.3. Preparation of Electroporation Competent Cells and Transformation of the Yeast Library 0.2-cm Gene Pulser/MicroPulser Cuvettes (Bio-Rad). Gene Pulser (Bio-Rad). Yeast strain EBY100. YPD medium: dissolve 20 g dextrose, 20 g peptone, and 10 g yeast extract in deionized H2O to a volume of 1 L and sterilize by filtration. This medium can be stored for 2 months at 4C. Lithium acetate (LiAc) and dithiothreitol (DTT) solution: dissolve 6.6 g LiAc and 1.54 g DTT in 1 L H2O. 1 M sorbitol solution: dissolve 182 g sorbitol in 1 L H2O. 1 M CaCl2 solution: dissolve 111 g in 1 L H2O. Electroporation buffer: add 1 ml of 1 1 M CaCl2 solution into 1 L of 1 1 M sorbital solution. SDCAA medium: dissolve 20 g dextrose, 6.7.
