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Overexpression of human processing alpha-glucosidase I and functional study of yeast processing alpha-glucosidase I

University of British Columbia

Processing α-glucosidase I (Glu I) holds a key regulatory position in the N-glycosylation pathway. Glu I mutations in humans have severe or fatal outcomes. Despite the important role of human Glu I (MOGS), very little information is available regarding the function, mechanism and structure of the enzyme. The lack of methods to produce sufficient quantities of the enzyme is a major drawback in carrying out further structural and mechanistic studies. Therefore, this thesis was focused on establishing a Pichia pastoris expression system for MOGS. MOGS with an N-terminal histidine (6xHis) tag was overexpressed in P. pastoris and purified by FPLC using a His-trap column. The purified MOGS was N-linked glycosylated and was enzymatically active (specific activity of 735 U/mg) against the synthetic substrate α-D-Glc1,2α-D-Glc1,3α-D-Glc–OCH₃. However, the overexpression of MOGS in P. pastoris was not reproducible and it was overexpressed intracellularly in subsequent expression trials. Therefore, overexpression of MOGS in E. coli was attempted, but the expressed MOGS accumulated as inclusion bodies with either a MBP (maltose binding protein) tag or a 6xHis-tag. Two methods (urea denaturation-refolding and freeze-thawing) were used to purify MOGS from inclusion bodies. However, less than 0.5 mg from one litre culture was recovered and it was not enzymatically active. As the overexpression of active MOGS in either P. pastoris or E. coli was not successful, further characterization of Glu I was continued with the study of the N-terminal function of Saccharomyces cerevisiae Glu I (Cwh41p). The Cwh41p clones with N-terminal truncations were constructed and the proteins were overexpressed in E. coli. Among the six truncations, five truncations (Cwh41Δ1-167p, Cwh41Δ1-93p, Cwh41Δ1-74p, Cwh41Δ1-54p and Cwh41Δ1-45p) were expressed mainly in inclusion bodies and the eluted fractions from His-trap column were not enzymatically active. However, the eluted fractions of Cwh41Δ1-39p and the internal control (Cwh41Δ1-34p) were active with a specific activity of 266 U/mg and 2000 U/mg, respectively. This indicates that the residues 35-45 of Cwh41p may be essential for the proper folding of Cwh41p. I propose that the α-helix (NH1) of Cwh41p plays a positive role in protein folding by interacting with C-domain of the enzyme.

Text

2016-09-06

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/59087

Food Science

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/