PRODUCTION OF Staphylococcal PROTEIN A IN E. coli: FED-BATCH FERMENTATION, PURIFICATION AND INMUNOCHEMICAL APPLICATION

KIKOT, PAMELA ALEJANDRA; VELASQUES ESPINOZA, LESLY; SOTO ESPINOZA, SILVIA; FLORES, CONSTANZA; GRASSELLI, MARIANO

CHAPADMALAL

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiología General; 2023

Sociedad Argentina de Microbiología General

Staphylococcus aureus protein A (SpA) is a single polypeptide protein of 42 kDa with a high affinity for Fc region of immunoglobulins (IgG). SpA is the preferred ligand for binding antibodies and molecules tagged with an Fc region in several immunological and biotechnological applications, such as affinity chromatography and immunochemical techniques. Therefore, there is a need for high-level production of the protein. SpA can be obtained through the culture of wild-type S. aureus and as a recombinant protein. This study focused on high-level fed-batch fermentative expression in E. coli of an engineered SpA-based ligand, AviPure [1]. This recombinant protein was used in two different applications. On the one hand, chromatographic resins were developed based on a commercial matrix for IgG purification from plasma. On the other hand, AviPure was used for oriented IgG-decoration of gold nanoparticles (Au-NPs) for diagnostic systems.Though SpA has five domains with an affinity for the Fc region, the molecule is incapable of binding five IgG molecules due to steric hindrance. This problem was overcome by using AviPure, which has a lower molecular weight (14 kDa) and contains two SpA domains, a histidine tag at the N-terminal for Ni–IDA-based purification and a Cys–His-Cys-His tag at the C-terminal for an oriented immobilization in solid supports [2]. Fed-batch cultures of E. coli BL21(DE3) harboring plasmid pET-28a(+) were conducted in a 5 L stirred tank bioreactor (BIOSTAT Aplus, Sartorius) with 20 g/L mineral medium with glucose [3]. Cell disruption was done in a high-pressure homogenizer (Panda 2K, GEA). Ni-IDA resin (IMAC sepharose FF, Cytiva) coupled to AKTA pure system was used to purify. The obtained protein was conditioned by gel filtration (Sepharose G-25, Cytiva) and lyophilized. Fed-batch culture of E. coli performed 5 L with 70 g dry cell weight per liter (179 DO600). The purification process yields 3.4 g of pure protein. AviPure was immobilized in Eupergit C via thiol-epoxy reaction and tested for Ig purification. Additionaly, Avipure were used to decore Au-NPs for biosensor applications. Site-specific immobilization of AviPure allows the Ig to keep oriented and retain their function. Au-NPs functionalization was followed throughout size increase by DLS and the shift of plasmon signal by UV-vis spectroscopy.[1] Kangwa et al (2015). AMB expr 5 (70).[2] Kikot et al (2014). J Mol Recognit 27(11). 659-68.[3] Riesenberg et al (1990). Appl Microbiol Biotechnol (34) 77-82