Caracterização biofísica da Frutalina recombinante e sua aplicação na purificação de Imunoglobulinas a1 (siga1) secretoras humanas

Abstract

Frutalin is a -D-galactose and -D-manose binding multilectin from Artocarpus incisa L. seeds. It belongs to the 'jacalin lectins related' family and presents around 98% identity with jacalina. Due to the high structural identity between jacalin and frutalin and the use of jacalin for IgA1 purification, a recent study showed the use of immobilized frutalin for human IgA1 purification. A low-cost alternative to the commercial ImmobilizedJacalin® collumn. One of the problems inherent in the use of immobilized frutalin as an alternative to human IgA1 purification is the amount of biological material necessary for the construction of chromatographic matrices. An alternative is the use of protein heterologously produced in microorganisms. This work aimed to biophysical characterization of recombinant frutalin (rFrutalin) and evaluation of its use as input in IgA1 purification of human colostrum. Frutalin was expressed in Pichia pastoris KM71H strain transformed and purified in agarose-Dgalactose columns (Sigma). For biophysical characterization circular dichroism (CD) analysis was performed to evaluate the thermal stability, influences of pH variation and prediction of secondary structures, and intrinsic fluorimetric analysis, dynamic light scattering (DLS) and scattering electrophoretic light (ELS or Zeta Potential). For the production of an IgA1 purification matrix of human colostrum, samples were tested in the rFrutalina (at concentration of 4 mg / ml matrix) in the locking process in the activated agarose. Then the samples were added to the activated matrices and maintained under stirring at 4 ° C for 2 minutes. Immobilization was evaluated at different times (2, 6 and 12 hours). The rFrutalina CD results showed a negative band with a maximum amplitude 218 and another positive band with a maximum 222 nm profile, similar to the native frutalin, showing predominantly sheet-β structure. Frutalin is stable, conformationally under acidic conditions at temperatures up to 60 ° C. The dynamic light scattering method (DLS) confirmed that the oligomerization of rFrutalina does not change with pH change and that its mass corresponds to approximately 59 ± 4 kDa. The agarose-frutalin matrices produced were able to retain a rich fraction IgA1 (FrIgA1). All tested matrices showed two chromatographic peaks: a peak related to proteins which did not interact with the matrix and another peak related to FrIgA1 that interacted with the matrix and was eluted with Dgalactose. Based on this result, it became clear that the downtime of the rFrutalin matrix influenced proportionally in its binder holding capacity, since the matrixsubjected to immobilization for 12 h showed better chromatographic performance. By the biophysical point of view, rFrutalina expressed in P. pastoris, showed no significant change when related to biophysical characteristics of the native frutalin, which supports the use of rFrutalina in IgA1 purification of human colostrum, as a viable alternative to commercial ImmobilizedJacalin® column.