An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression inEscherichia colicombined with intein-mediated protein ligation

Abstract

In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications.

Production of foot-and-mouth disease virus SAT2 VP1 protein

“…2). Other researchers have also reported recombinant protein yield increases when using EnPresso ® B compared to LB broth (Ta et al 2015;Zarschler et al 2013). The ratio of protein yield per cell was, however, similar for EnPresso ® B medium and LB broth.…”

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference

“…Recombinant expression of VHH can produce single domain antibody (sdAb) with dimensions in the nanometer range, which is also known as nanobody (Nb). High expression yield of Nbs can be obtained in various expression systems, such as bacteria, fungi and plants [10–12]. …”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…This strategy became available 30 years ago, applied to different recombinant proteins which require disulfide bonds to fold into their native structure and progressively improved with the introduction of more efficient strains such as Rosetta-gami B (DE3) or SHuffle T7 cells. These have been used for the cytoplasmic production of naked nanobodies [110,111] and of their fusion variants containing at their Cterminus an intein-chitin domain suitable for site-specific alkyne functionalization that requires reducing conditions for its functionality [109,112,113]. In one case it was confirmed that the binding properties of the non-modified nanobodies produced in the periplasm were preserved when the same binders were expressed as fusion reagents in the cytoplasm of Shuffle T7 cells [109].…”