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 language | English |
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Pages (from-to) | 351-363 |
Number of pages | 13 |
Journal | Protein Engineering Design & Selection |
Volume | 28 |
Issue number | 10 |
DOIs | |
Publication status | Published - 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