Direct capture and selective elution of a secreted polyglutamate‐tagged nanobody using bare magnetic nanoparticles

Abstract

Background The secretion and direct capture of proteins from the extracellular medium is a promising approach for purification, thus enabling integrated bioprocesses. Major Results We demonstrate the secretion of a nanobody (VHH) to the extracellular medium (EM) and its direct capture by bare, non‐functionalized magnetic nanoparticles (MNPs). An ompA signal peptide for periplasmic localization, a polyglutamate‐tag (E8) for selective MNP binding, and a factor Xa protease cleavage site were fused N‐terminally to the nanobody. The extracellular production of the E8‐VHH (36 mg L–1) was enabled using a growth‐decoupled Escherichia coli‐based expression system. The direct binding of E8‐VHH to the bare magnetic nanoparticles was possible and could be drastically improved up to a yield of 88% by adding polyethylene glycol (PEG). The selectivity of the polyglutamate‐tag enabled a selective elution of the E8‐VHH from the bare MNPs while raising the concentration factor (5x) and purification factor (4x) significantly. Conclusion Our studies clearly show that the unique combination of a growth‐decoupled E. coli secretion system, the polyglutamate affinity tag, non‐functionalized magnetic nanoparticles, and affinity magnetic precipitation is an innovative and novel way to capture and concentrate nanobodies.

Recombinant protein purification and immobilization strategies based on peptides with dual affinity to iron oxide and silica

“…[ 11 ] The same buffering system was also used to successfully elute a Glu6‐tagged GFP from BIONs. [ 3,10 ] This effect has been ascribed to the high affinity of phosphate anions to iron oxide, which bind to iron oxide via surface complex formation, thus competing with the peptide tag binding and promoting the desorption of the tagged protein. [ 3,10,11 ] Here, PBS (pH 7.5) also promoted the elution of all tagged EGFPs (Figure 1A).…”

“…[ 2,3,10 ] Thus, the use of bare, non‐functionalized iron oxide and silica surfaces allows saving time, reducing costs, and the ecological footprint. [ 7,10 ]…”

“…Recombinant protein purification and immobilization strategies based on unmodified iron oxide and silica matrices make use of the affinity of solid‐binding peptides to iron oxide [ 3,10–12 ] or silica [ 6,7,13–17 ] to promote the selective binding of the desired protein(s). Iron oxide‐ and silica‐binding peptides can be readily fused to proteins via recombinant DNA technology, typically at their N‐ or C‐terminal, allowing for high‐affinity and site‐directed immobilization onto unmodified iron oxide or silica surfaces.…”

“…Solid‐binding peptides consisting of repeating glutamate (Glu6/Glu8), histidine (His6 and (HR)4/(RH)4) or arginine ((HR)4/(RH)4) subunits were shown to display high affinity towards bare iron oxide nanoparticles (BIONs) and to work effectively as iron oxide affinity tags in protein purification/immobilization. [ 3,10–12 ] These charged amino acids interact with iron oxide surfaces mainly via ionic and coordinate bonding. [ 9,20,21 ] Arginine‐ (e.g., CotB1/CotB1p/SB7, (HR)4/(RH)4) and EctP1), lysine‐ (e.g., Car9) and histidine‐rich (His6, (HR)4/(RH)4 an EctP1) peptides display high affinity towards bare silicon dioxide surfaces and have been effectively used as affinity tags for silica‐based materials in protein purification/immobilization.…”

From spectral analysis to hysteresis loops: a breakthrough in the optimization of magnetic nanomaterials for bioapplications

“…The last two decades have witnessed a boost of biomedical applications based on the unique properties of magnetic nanomaterials [1][2][3][4]. Magnetic single core [5] and multi-core nanoparticles [6], either bare [7] or submitted to surface functionalization [8,9], core-shell systems [10,11], supraparticles [12], hollow nanoparticles [13], nanochains [14,15] and nanodiscs [16,17] are being actively investigated in view of their application in modern therapies of precision medicine [18,19] as well as in radiation-free, non-invasive imaging or spectroscopy techniques and in the development of sensitive bionsensors [20][21][22]. A common feature of most of the above mentioned applications is that they typically make use of magnetic nanomaterials driven at high frequency in the non-linear magnetization regime [23] (the strictly linear magnetization regime, i.e.…”