Expression, purification, and analyses of glycosylation and disulfide bonds of Stereum purpureum endopolygalacturonase I in Pichia pastoris.
We have succeeded in the expression of Stereum purpureum endopolygalacturonase I (EndoPG I) using the Pichia expression system and in purification of the three kinds of recombinant EndoPG I, which have one to three sugar chains by using CM52 column chromatography. The sugar chains which were added to EndoPG I were the M8, M9, and/or M10 high-mannose type. The results of LC-MS analysis showed that recombinant EndoPG Is were randomly glycosylated at four N-glycosylation sites. From the thermal denaturation curves of the recombinant enzymes, it was suggested that EndoPG I differing in thermal stability was included in the sample after purification. Therefore, we investigated the disulfide bonds of recombinant EndoPG I by LC-MS analysis. As a result, peptides without a second or third disulfide bond were detected. This result is the first indicating that there are incomplete enzymes in terms of disulfide bonds in the Pichia expression system.
Ogawa
Shun
S
Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Aomori Prefecture, Hirosaki 036-8561, Japan.
Shimizu
Tetsuya
T
Ohki
Hajime
H
Araya
Tomoyuki
T
Okuno
Toshikatsu
T
Miyairi
Kazuo
K
D000363:Agaricales / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006031:Glycosylation; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011096:Polygalacturonase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D004220:Disulfides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D011096:Polygalacturonase