Fig. 2.

Overview of the hBChE library construction. The mutation library construction was divided into four stages. Stage 1, both site-saturation mutagenesis (library 0) and randomized mutagenesis (libraries 1–6) were done through PCR using designed primers. Stage 2, PCR products were then cloned into a pENTR1A vector to establish a mutation library. Twenty clones from the library were randomly picked and sequenced to verify the diversity of the library (Table 1). Stage 3, the mutation library in pENTR1A plasmid was then exchanged to destination vector pAD/CMV-DEST to establish an adenoviral library by conducting a clonase reaction. Stage 4, after transfection of HEK 293A cells with a pAD library, the adenovirus library stock was prepared after packaging in HEK 293A cells.