Optimizing the expression of an anti-Leishmania nanobody ‘ALNb18’ produced free or fused with super folder GFP

Abstract

Nanobodies belong to a new generation of antibodies produced by recombinant engineering and have high perspectives in biopharmaceutical drug formulations. In this work, we tested a new nanobody (ALNb18) against Leishmania, a parasitic intracellular pathogen, and optimized various conditions to enhance its expression yield in Escherichia coli. Many bacterial growth conditions were optimized for better expression, including host cell, inducer concentration, medium, pre-induction growth, incubation temperature and incubation duration. Nanobody expression levels were compared between these conditions using enzyme-linked immunosorbent assay (ELISA) against immobilized leishmania antigens. As an alternative strategy, nanobody ALNb18 was produced fused with super-folder green fluorescent protein (sfGFP) to enhance its yield of expression. The nanobody levels of expression were approximately the same regardless of the host E. coli strain with superiority to WK6 strain in time saving. Using LB medium, 0.1 mmol/L of expression inducer isopropyl β-d-1-thiogalactopyranoside (IPTG) and incubation at 37 °C for only 4 h showed better nanobody expression. Adding sfGFP downstream the sequence of ALNb18 resulted in a fully antigen-reactive fusion protein that was recovered from the periplasm of E. coli, with a slight increase in expression levels. Recombinant ALNb18 could be collected and purified in its effective form in quantities up to a milligram per litter of liquid cultures in flask. Affordable, time saving and economical methods were optimized to enhance the expression yield of ALNb18 nanobody, including its fusion to sfGFP. This nanobody could be a promising tool for ensuing pharmaceutical studies with a particular emphasis on leishmania infection.

Keywords

• Nanobody
• expression optimization
• Leishmania
• sfGFP