KONSTRUKSI pET-16b_rec DENGAN METODE MUTAGENESIS SITUS TERARAH SEBAGAI VEKTOR EKSPRESI UNTUK PROTEIN FUSI POLIHISTIDIN DI UJUNG KARBOKSI PADA SISTEM ESCHERICHIA COLI

• Main Author: Liddini Hanifa, Hanina
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44649
• Institution: Institut Teknologi Bandung

One of the most frequently methods used to purify recombinant proteins is affinity column chromatography. Recombinant proteins can be efficiently purified by nickel affinity column when fused with polyhistidine residue on either N-terminal or C-terminal of proteins. Position of polyhistidine tags depends on the active site or important domain of protein. Hence, a suitable expression vector is needed. pET-16b is a vector expression which enables fusion of polyhistidine tags (His-tag) with recombinant protein on N-terminal. The objective of this experiment was to reconstruct pET-16b by PCR-based Site Directed Mutagenesis (PCR-based SDM) to generate new plasmid called pET-16b_rec. This plasmid enables fusion of His-tag and recombinant protein on C-terminal. In this study, primer annealing temperature, annealing time, extension time of PCR, and type of DNA polymerase were optimized to obtain optimal products of pET-16b_rec. PCR-based SDM products were digested with DpnI 10 U, ligated, and transformed into E. coli TOP10. Potential plasmids obtained after transformation were characterized and sequenced. The unsuitable sequences of pET-16b_rec were corrected by SDM-SPRINP method. The products of this method were also digested with DpnI 30 U, transformed into E. coli TOP10, and sequenced. The optimum primer annealing condition for PCR-based SDM was 10 seconds at 68 °C, while the optimum extension time was 4 minutes. Q5 HiFi Polymerase gave the most desired product yield than other two enzymes. The optimal primer annealing temperature for SDM-SPRINP was 60 °C with 30 times PCR cycles. In conclusion, pET-16b_rec was successfully constructed with the size of 5684 bp and it has correct nucleotide sequences.