Selecting subpopulations of high-quality protein conformers among conformational mixtures of recombinant bobine MMP-9 solubilized from inclusion bodies

JOSE VICENTE CARRATALA; LAIA GIFRE-RENOM,; ANTONIO VILLAVERDE; ANNA ARIS; ELENA GARCIA-FRUITÓS; JULIETA M. SANCHEZ; NEUS FERRER MIRALLES

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI

Lugar: Basel; Año: 2021

1422-0067

A detailed workflow to analyze the physico-chemical characteristics of mammalian matrix met-alloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and charac-terized. Therefore, with the aim to link the physico-chemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the sepa-ration of subpopulations of conformers with specific characteristics. In protein purification pro-cedures, the detailed analysis of the individual physico-chemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of infor-mation to select for the best-fitted protein populations.