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

Duy Tien Ta, Erik Steen Redeker, Brecht Billen, Gunter Reekmans, Josephine Sikulu, Jean-Paul Noben, Wanda Guedens, Peter Adriaensens*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

23 Citations (Web of Science)

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(A (R)) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His(6) 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 similar to 45 mg/l in the EnPresso(A (R)) 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.

Original languageEnglish
Pages (from-to)351-363
Number of pages13
JournalProtein Engineering Design & Selection
Volume28
Issue number10
DOIs
Publication statusPublished - Oct 2015

Keywords

  • bioorthogonal chemistry
  • CuAAC
  • cytoplasmic expression
  • intein-mediated protein ligation
  • VCAM1-targeting nanobody
  • SINGLE-DOMAIN ANTIBODIES
  • CELL-ADHESION MOLECULE-1
  • RECOMBINANT EXPRESSION
  • CHEMICAL LIGATION
  • PURIFICATION
  • CANCER
  • IMMOBILIZATION
  • FRAGMENTS
  • IMMUNOGLOBULINS
  • BIOCONJUGATION

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