Analysis of recombinant human saposin A expressed by Pichia pastoris.
Saposins (SAPs) are small glycoproteins required for activation of sphingolipid hydrolysis by lysosomal enzymes. Four SAPs, SAP-A, -B, -C, and -D, are proteolytically cleaved from a single gene product termed prosaposin. The mature coding sequence of human SAP-A tagged with 6-histidine was expressed in Pichia pastoris and the recombinant protein was purified from the culture supernatant by simple purification steps with an immobilized metal ion affinity column, a Concanavalin A column, and reversed-phase HPLC. Secreted SAP-A contained both glycosylated and nonglycosylated forms. Both forms of SAP-A activated galactocerebroside and 4-methylumbelliferyl beta-d-glucoside hydrolysis by galactocerebrosidase and glucocerebrosidase. SAP-A expressed in P. pastoris should be useful for further structural and functional analysis of this protein.
Yamada
Minoru
M
Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, Murodo-cho, Izumi, Japan.
Inui
Koji
K
Hamada
Daizo
D
Nakahira
Kumiko
K
Yanagihara
Keiko
K
Sakai
Norio
N
Nishigaki
Toshinori
T
Ozono
Keiichi
K
Yanagihara
Itaru
I
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005698:Galactosylceramidase / Q000378:metabolism; D015870:Gene Expression; D005796:Genes / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D049231:Saposins
D006023:Glycoproteins; D011994:Recombinant Proteins; D049231:Saposins; D005698:Galactosylceramidase