Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris

Abstract

BackgroundProprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications. However, it is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides.ResultsIn this study, we evaluated the ability of P. pastoris to process furin cleavage sites and how to improve the cleavage efficiencies of furin cleavage sites in P. pastoris. The results showed that P. pastoris can process furin cleavage sites but the cleavage efficiencies are not high. Arg residue at position P1 or P4 in furin cleavage sites significantly affect cleavage efficiency in P. pastoris. Kex2 protease, but not YPS1, in P. pastoris is responsible for processing furin cleavage sites. Heterologous expression of furin or overexpression of Kex2 in P. pastoris effectively increased cleavage efficiencies of furin cleavage sites.ConclusionsOur investigation on the processing of furin cleavage sites provides important information for recombinant expression of furin precursors in P. pastoris. Furin or Kex2 overexpressing strains may be good choices for expressing precursors processed by furin in P. pastoris.

Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System

“…However, it was observed that the glucanase enzyme with a theoretical molecular weight of approximately 34 kDa was produced in 2 bands, approximately 34 and 38 kDa. A similar situation was reported in the study by Huang et al (2018) and they showed that GFP (Green fluorescence protein) production was seen as 2 protein bands in SDS-PAGE analysis. They explained that the observation of the same protein in two bands of different sizes may be due to the 9 kDa signal sequence remaining unprocessed during extracellular secretion.…”

Secretory expression of β-1,3-glucomannanase in the oleaginous yeast Rhodosporidium toruloides for improved lipid extraction

“…It should be noted that similar phenomena have been demonstrated for secretory expression of green fluorescent protein in Pichia pastoris (Huang et al 2018).…”

Bioinformatic analysis of the spike protein cleavage sites of coronaviruses in the mammalian order Eulipotyphla

“…Furins are ubiquitously expressed in various sites in all cells and tissues in eukaryotes, but little is known about whether the difference in furins across different species might affect their ability or efficiency to cleave the coronavirus spike protein (Hoffmann et al, 2018). Hence, more animals should be included, and more experiments need to be carried out to draw the conclusions (El Najjar et al, 2015; Huang et al, 2018).…”