Example 6
Each plasmid containing EBP, CalM, or a Cys-incorporated block was used to prepare genes for the fusion polypeptide libraries of EBP[G1A3F2]n-CalM-EBP[G1A3F2]n with Cys blocks at both ends. A plasmid vector harboring an EBP gene was digested and dephosphorylated with 10 U of XbaI, 10 U of BseRI and 10 U of FastAP, a thermosensitive alkaline phosphatase, in CutSmart buffer for 30 minutes at 37° C. The restricted plasmid DNA was purified using a PCR purification kit, and then was eluted in 40 μl of distilled and deionized water. A total of 4 μg of the CalM gene was digested with 10 U of XbaI and 15 U of AcuI in the CutSmart buffer for 30 minutes at 37° C. to prepare the CalM gene as an insert, followed by separation using agarose gel electrophoresis and purification using a gel extraction kit. A ligation reaction was performed by incubating 90 pmol of the purified insert with 30 pmol of the linearized vector in T4 DNA ligase buffer containing 1 U of T4 DNA ligase for 30 minutes at 16° C. The product was transformed into Top10 chemically competent cells, then the cells were plated on a SOC plate supplemented with 50 μg/ml of ampicillin. Transformants were initially screened by a diagnostic restriction digest on an agarose gel and further confirmed by DNA sequencing as described above.
