Magnetic Nanobeads technology for fast and efficient protein purification

NAHUEL A. RAMELLA; GOTELLI, GUSTAVO; M ALEJANDRA TRICERRI; ROSU, S. A

Rosario

Encuentro; L Reunión Anual Sociedad Argentina de Biofísica; 2022

SAB

Protein purification is a critical limiting step in the development of some biological experimental design. Depending on the different protein chemical and physical properties, each protocol must be adapted in order to obtain the best compromise involving lower time and protein aggregation but showing the highest purity and yield. Within this area, a commonly used principle of operation is IMAC affinity chromatography, based on selective complex formation between proteins and transition metals as Nickel, Copper, Zinc, Cobalt or Manganese. This affinity process applies to some natural proteins and others whose amino-acid sequence was biotechnology modified and may be expressed in several systems. In case of transgenic proteins and since years ago, it was designed the technology of N or C terminal modified proteins constructs containing a His-Tag which strengthens the metal ion affinity of the whole protein. Nevertheless, in bothcases IMAC allowed the specific retention and purification of proteins with metal affinities from complex biological mixtures. However, that experimental design involves high costs and long-time processing. Here we show the technology of coated magnetic nanoparticles, which in a short time and with high efficiency allows to purify proteins based in IMAC principle. Magnetic beads in the nanometerrange (core around 20 nm) were coated with a polymer shell containing Imino-Diacetic-Acid which is a tridentate metal chelator. His-Tagged Apolipoprotein A-I was added in a low-ionic strength buffer at physiological pH for five minutes and then stand in a magnetic separator. Supernatant was separated, beads were washed in a regular buffer and following 0.5 M imidazole buffer was added in order to separate bound protein. Altogether, the purification step was complete withing 30 minutes. This procedure was proved to be effective even starting from the  E.coli lysate broth with minor modifications in comparison with the usual column protocols. The relative efficiency was about 23 mg protein/gr beads, and the total time of purification was about 2 h. We conclude that this technology offers a fast and efficient procedure for protein purification, thus preserving proteins from structural disorganization.  We are now working to apply this technology on the purification of plants proteins of interest like lectins, some of which have natural affinity for transition metals without need to add His-tag modifications, allowing its direct purification. Acknowldegments Acknowledgments: INIBIOLP, CCT-La Plata, CONICET, Facultad de Ciencias Médicas, UNLP