EKSPRESI HALF-ANTIBODY ANTI-PSTS1 PADA E. COLI ORIGAMI B (DE3) DENGAN VARIASI SUHU DAN WAKTU INDUKSI SEBAGAI PENGEMBANGAN AWAL KIT DIAGNOSTIK TUBERKULOSIS BERBASIS ANTIBODI BISPESIFIK

• Main Author: Kevin Setiawan, Gregorius
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/84688
• Institution: Institut Teknologi Bandung

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis and is the second leading cause of mortality due to a single infectious agent, following Covid-19. To date, most of the diagnostic methods available are only capable of detecting active TB. Meanwhile, diagnostic methods for detecting latent TB still face challenges such as false positive results and limited access to diagnostic tools in peripheral areas. Bispecific antibody-based diagnostic methods have the potential to detect both active and latent TB simultaneously. PstS1 is an antigen that has been proven to be a target for detecting latent TB. However, research and development of antibodies targeting the PstS1 antigen have never been conducted. This research aims to optimize the expression of anti-PstS1 half-antibody in E. coli Origami B (DE3) with variations in temperature and induction time as part of the development of bispecific antibodies. The synthetic gene encoding the light and heavy chains of anti-PstS1 was transformed into E. coli Origami B (DE3) and subsequently confirmed by colony PCR. Plasmid and total RNA were isolated from the transformed bacterial colonies grown in Luria Bertani liquid medium. The plasmid isolates were then sequenced, and the DNA sequences were analyzed. Total RNA from the recombinant bacterial culture was isolated, followed by RT-PCR to confirm the transcription of the target gene. Secondary structure evaluation of mRNA was performed using RNAfold and UNAFold, utilizing the Minimum Free Energy and Centroid approaches. Anti-PstS1 expression was conducted with variations in induction temperature (37?, room temperature, and 16?) and induction time (during early, mid, and late logarithmic phases) with the addition of IPTG. The expression results from the soluble and insoluble fractions were purified using a Ni-NTA column. Based on the results obtained, it was found that plasmid and total RNA were successfully isolated. Sequencing results showed the presence of missense substitutions and insertions in one of the two sequencing results, forming a premature stop codon in the heavy chain and producing a 37.45 kDa protein. RNA isolation and RT-PCR results indicated successful transcription of the light and heavy chains. Secondary structure evaluation of mRNA showed the formation of a secondary structure at the Ribosome Binding Site (RBS) of the light chain, which was more favorable compared to the heavy chain RBS. In the visualization of SDS-PAGE results from protein expression, no bands of approximately 23 kDa (light chain) and 55 kDa (heavy chain) were observed. However, bands of approximately 37 kDa were observed at 37?, room temperature, and 16? in the soluble fraction during the early and mid-logarithmic growth phases, and in the insoluble fraction during the early, mid, and late logarithmic growth phases. The conclusion drawn from this study is that the heavy chain of the anti-PstS1 half-antibody was successfully expressed in a truncated form, but the light chain of the anti-PstS1 half-antibody was not expressed. Therefore, further research is needed to achieve better recombinant protein expression.