Identification and characterization of N-glycosylation site on a Mucor circinelloides aspartic protease expressed in Pichia pastoris: effect on secretion, activity and thermo-stability

Abstract

Methylotrophic yeasts have widely been used as model organisms for understanding cellular functions and biochemical activities in lower eukaryotes. The gene encoding an aspartic protease (MCAP) from Mucor circinelloides DSM 2183 was cloned and expressed into Pichia pastoris using both the native M. circinelloides signal peptide (mcSP) and α-factor secretion signal from Saccharomyces cerevisiae (α-MF). When expressed in P. pastoris using α-MF and mcSP, MCAP was secreted into the culture medium at a concentration 200 mg L−1 (410 MCU mL−1) and 110 mg L−1 (249 MCU mL−1), respectively. The SDS-PAGE analysis of each culture shows that the protein was secreted in the media in two forms with molecular weights of approximately 33 and 37 kDa. Upon digestion using endoglycosidase H (Endo H), only one band at 33 kDa was observed, indicating that the protein might be glycosylated. One putative N-glycosylation site was found and a site-directed mutagenesis at position Asn331-Gln of the sequence produce only one form of the protein of 33 kDa, similar to that obtained when digested with Endo H. The optimum temperature and pH activity of the expressed MCAP was found to be at 60 °C and 3.6, respectively.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0691-3) contains supplementary material, which is available to authorized users.

Cloning and Expression of an Active Aspartic Proteinase Gene From Aspergillus Oryzae DRDFS13 in Pichia Pastoris

“…E. coli strains were grown overnight in Luria-Bertani medium (10 g L -1 , tryptone, 5 g L -1 yeast extract, 5 g L -1 NaCl) at 37 °C, 220 rpm. P. pastoris X-33 was grown in YPD medium (10 g L -1 yeast extract, 20 g L -1 peptone, 20 g L -1 glucose) at 30 °C for 3 days with shaking at 250 rpm (Kangwa et al 2018).…”

“…Electrophoresis was run for 50 min at 300 V and 60 mA. The gel was then stained with Coomassie Brilliant Blue (AppliChem TM) overnight and then detained overnight with the distaining solution (Kangwa et al, 2018).…”

“…The ampli ed aspartic protease gene (fragment) had a size of 1.2 kbp (Fig.3.1). Similarly, the gene encoding milk-clotting aspartic protease (MCAP) from M. circinelloides strain DSM 2183 (Antonio et al, 2013), MCAP protein from M. circinelloides DSM 2183 (Kangwa et al 2018), yak chymosin from Yak (Luo et al 2016) and aspartic proteinase from M. mucedo DSM 809 (Yegin and Fernandez-Lahore, 2013) were 959 bp, 1229 bp, 1,008 bp and 1,200 bp long, respectively.…”

“…Antonio et al, (2013), reported the expression of the aspartic protein gene from M. circinelloides in P. pastoris under the control of the constitutive GAP promoter. Similarly, the gene encoding an aspartic protease (MCAP) from M. circinelloides DSM 2183 cloned in P. pastoris was successfully expressed using both the native M. circinelloides signal peptide (mcSP) and α-factor secretion signal from Saccharomyces cerevisiae (α-MF) (Kangwa et al 2018).…”

“…The methylotrophic yeast Pichia pastoris, currently reclassi ed as Komagataella pastoris, has become a substantial workhorse for biotechnology, especially for heterologous protein production (Ahmad et al, 2014). P. pastoris have many advantages in yielding a high-level expression of recombinant proteins, protein processing and are characterized by post-translational modi cations (Kangwa et al, 2018;Luo et al, 2016). The post-translation modi cation associated with higher eukaryotes such as processing of signal sequence, folding, disul de bridge formation, certain types of lipid addition and Oand N-linked glycosylation (Cereghino and Cregg 2000).…”