Generation and characterization of some UnaG mutants | Abstract
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Journal of Computational Methods in Molecular Design


Generation and characterization of some UnaG mutants

Author(s): Numan Eczacioglu

UnaG protein from Japan eel (Anguilla japonica) is a novel fluorescent protein with binding domain that acquires fluorescence when bound to unconjugated bilirubin (UC-BR). In this study, several point mutations (F17M, N57D, N57E, N57R, L41F, Y99F_Y134W, Y99M_Y134M, and W9F_W103F) were made on the UnaG nucleotide sequence via using a method for sequence and ligation independent cloning (SLIC). The aim of the mutations on UnaG is to figure out the change in fluorescence properties. The new mutagenic vector was transformed into the commercial competent cells (E. coli Mach1) by using heat shock at 42 °C for 2 minutes. Transformed cells were grown on and selected from the LB agar plate with ampicillin. (1:1000). The DNA sequencing results show that all these mutations have done correctly. The expression of the mutant proteins was made in the pTOLT expression system by inducing with IPTG. Cells were collected with high speed centrifugation