A novel N-terminal region of the membrane beta-hexosyltransferase: its role in secretion of soluble protein by Pichia pastoris

作者：Dagher Suzanne F; Bruno Barcena Jose M^*

来源：Microbiology-SGM, 2016, 162(1): 23-34.

DOI：10.1099/mic.0.000211

The beta-hexosyltransferase (BHT) from Sporobolomyces singularis is a membrane-bound enzyme that catalyses transgalactosylation reactions to synthesize galacto-oligosaccharides (GOSs). To increase the secretion of the active soluble version of this protein, we examined the uncharacterized novel N-terminal region (amino acids 1-110), which included two predicted endogenous structural domains. The first domain (amino acids 1-22) may act as a classical leader while a non-classical signal was located within the remaining region (amino acids 23-110). A functional analysis of these domains was performed by evaluating the amounts of the rBHT forms secreted by recombinant P. pastoris strains carrying combinations of the predicted structural domains and the a mating factor (MF alpha) from Saccharomyces cerevisiae as positive control. Upon replacement of the leader domain (amino acids 1-22) by MF alpha (MF alpha-rBht((23-594))), protein secretion increased and activity of both soluble and membrane-bound enzymes was improved 53- and 14-fold, respectively. Leader interference was demonstrated when MF alpha. preceded the putative classical rBHT((1-22)) leader (amino acids 1-22), explaining the limited secretion of soluble protein by P. pastoris (GS115 : : MF alpha-rBht((1-594)). To validate the role of the N-terminal domains in promoting protein secretion, we tested the domains using a non-secreted protein, the anti-beta-galactosidase single-chain variable antibody fragment scFv13R4. The recombinants carrying chimeras of the N-terminal 1-110 regions of rBHT preceding scFv13R4 correlated with the secretion strength of soluble protein observed with the rBHT recombinants. Finally, soluble bioactive HIS-tagged and non-tagged rBHT (purified to homogeneity) obtained from the most efficient recombinants (GS115 : : MF alpha-rBht((23-594))-HIS and GS115 : : MF alpha-rBh((23-594))) showed comparable activity rates of GOS generation.

• 出版日期2016-1