Rational design of Zeocin binding protein variants for antibiotic resistance studies

Abstract

Antibiotic resistance marker was used in this investigation because they are selected;e markers and effective in different hosts. This study used Zeocin binding protein (ZBP) due to its known 3D structure and applicability in prokaryotic and eukaryotic hosts. A library of 22 mutations was developed through a rational design strategy. Subsequently, a selection strategy was used to identify destabilizing mutations in the ZBP. ZBP variants were expressed in E.coli using a leaky expression approach, and minimum inhibitory concentration (MIC) was calculated by cultivating different variants at various Zeocin concentrations. Zeocin resistance was drastically decreased in some ZBP variants. Positive controls (wild-type ZBP) exhibited high resistance, while negative controls (pET vector without ZBP) showed susceptibility. Two variants (ZBP P9E and ZBP R26F) displayed drastic resistance loss. The variants reported in this study may identify molecular chaperones and folding modulators affecting proteostasis. These variants can potentially discover chemical chaperones that improve the stability and solubility of destabilized ZBP variants.

Keywords

• Bleomysin
• Zeocin
• Biosensor
• Folding