Nanobeads technology for fast and efficient protein purification

GOTELLI, G.; RAMELLA, NAHUEL A.; ROSÚ, S A.; TRICERRI, M. A.

Rosario, Santa Fe

Congreso; L Reunión Anual Sociedad Argentina de BiofísicaL Reunión Anual Sociedad Argentina de Biofísica. .; 2022

Sociedad Argentina de Biofísica.

Topic Area: New Technologies Title:Nanobeads technology for fast and efficient protein purificationGustavo Gotelli1; Nahuel Ramella2, Silvana A. Rosú2, María Alejandra Tricerri2; (1) Kalium Technologies, Investigación y Desarrollo, Zeballos 1716 Bernal, CP 1876, Buenos Aires, Argentina(2) INIBIOLP, CCT-La Plata, CONICET, Facultad de Ciencias Médicas, UNLP, 60 y 120, La PlataProtein purification is a critical limiting step in the development of all 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 aggression 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, Iron, Zinc, Cobalt and 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 as bacteria, yeast, fungi, plants or animal cells. 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 both cases 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 nanometer range (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 original bacterial pellet with minor modifications. 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.