CLONING OF SYNTHETIC INTERFERON ALFA-2B (IFNa2b) CODING REGION IN Escherichia coli, OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT IFNa2b PROTEIN

• Main Author: ASMANA NINGRUM (NIM 20706011); Pembimbing: Debbie Sofie Retnoningrum, Ph.D. dan Dr. Heni Rach, RATIH
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/13076
• Institution: Institut Teknologi Bandung

Interferon (IFN) is a cytokine produced and secreted by almost all eucaryotic cells as a response to viral, bacterial, antigen, or mitogen stimuli. Based on their receptor type on the cell membrane surface, IFN is classified to type I and type II. Type I consists of IFNα, IFNβ, IFN τ, and IFN ω. Tipe II consists of IFN γ. Hepatitis B and C are serious diseases because if not managed properly can develop hepatocellular carcinoma. WHO data in 2002 showed that the mortality worldwide is 1 million people per year for hepatitis B and 1.4 million people for hepatitis C. Up to now IFNα2b is a standard therapy for hepatitis B and hepatitis C both as a single therapy or in combination with other nucleosides analog. IFNα therapy for hepatitis B needs 48 weeks and 24 to 48 weeks for hepatitis C. The cost therapy with IFNα2b is quite high, 2.5 to 2.8 million per single dose and can not be achieved by all Hepatitis B or hepatitis C patients. This research was intended to obtain recombinant IFNα2b protein by cloning of synthetic IFNα2b coding region in Escherichia coli (E. coli). The nucleotide sequence of IFNα2b Open Reading Frame (ORF) has been known and its codons have been optimized for E. coli. The IFNα2b ORF was synthesized using 10 ligonucleotides by Thermodynamically Balanced Inside-Out (TBIO) method. Polymerase Chain Reaction (PCR) was used to amplify IFNα2b ORF and introduce the HindIII and EcoRI restriction sites. The ORF was then ligated to pGEM-T cloning vector and the ligation product was transformed into E. coli JM109. The nucleotide sequencing was conducted to obtain the nucleotides sequence of IFNα2b ORF in recombinant plasmids. Recombinant pGEM-T carrying IFNα2b ORF was obtained, however it contained a deletion mutation of deoxyguanocine monophosphate in second codon. The ORF was successfully inserted into pET32b expression vector and the ligation product was transformed into E. coli DH5α resulting pET32b IFNα2b G del recombinant plasmid. The site directed mutagenesis was done to repair the nucleotide sequence of IFNα2b ORF. The mutagenesis product was transformed into E. coli TOP10. pET32b IFNα2b containing the correct sequence was transformed into E. coli BL21. One transformant was induced by isopropyl β-D-1-tiogalactopiranoside (IPTG) to obtain IFNα2b recombinant protein. The protein product was a fusion protein with polihistidin to simplify the purification. The protein was characterized by Sodium Deodesyl Sulphate Poly Acrylamide Gel Electrophoresis (SDS PAGE) method, showed the recombinant IFNα2b band as a 37 kDa protein. The purification was conducted by nickel column and the purified protein was characterized by Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI TOF) mass spectrometry. The result showed that the recombinant protein have high homology to IFNα, which IFNα2b is one of its member.