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Recombinant production and structural studies of a serine peptidase from Scopulariopsis koningii
Author(s) -
Pedezzi Rafael,
Evangelista Danilo E.,
Polikarpov Igor,
Cabral Hamilton
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.467.1
Subject(s) - pichia pastoris , recombinant dna , in silico , biochemistry , serine , molecular mass , biology , enzyme , chemistry , gene , microbiology and biotechnology
Scopulariopsis koningii is an ascomycetes fungus with high biotechnological potential, due to its peptidase. Previous work showed high colagenolytic activity in S. koningii cultured in feather flour rich medium. A native serine peptidase was purified from this bioprocess and has been shown biotechnological potential. However, this fungus has a poor molecular data available in databanks. Thus, we proposed a transcriptomic study to better describing the S. koningii enzymatic arsenal and to find this serine peptidase gene. The gene was in silico selected and synthetized in pPICZα‐A vector to a Pichia pastoris recombinant production. The protein production and purification were carried out in methanol‐induced system followed by Ni‐affinity chromatography. A structural study was carried out by a 3D model construction, generated by homology, and SAXS data collection. The recombinant protein was produced with the pro‐region, which act as an activity inhibitor. The molecular weight of pro‐region plus catalytic region is 40 kDa. SAXS studies showed a globular monomeric protein with Rg ~ 22.5 Å and Dmax ~ 80.3 Å. N‐terminus region build a protuberance related to pro‐region. This region is flexible and, as has been shown elsewhere, acts as activity inhibitor in trans. This structural study has enlightened activation strategies that can bring more control of the peptide hydrolysis start. Support or Funding Information FAPESP Proc. n° 2016/20385‐2 and 2015/16084‐4 and CAPES. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .