Single-step purification by lectin affinity and deglycosylation analysis of recombinant human and porcine deoxyribonucleases I expressed in COS-7 cells.
Human and porcine recombinant deoxyribonucleases I (DNases I) were expressed in COS-7 cells, and purified by a single-step procedure. Since affinities for concanavalin A (Con A) and wheatgerm agglutinin (WGA) were strong in these recombinant DNases I, purification using Con A-WGA mixture-agarose column was performed. By this method, the enzymes in culture medium could quickly be isolated to apparent homogeneity in approx. 10 min. From 1 ml of culture medium, about 20-30 microg of purified DNase I with a specific activity ranging from 22000 to 41000 units/mg were obtained. The purified DNases I were subjected to enzymatic deglycosylation by either peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). The recombinant enzyme was cleaved by PNGase F, but not by Endo H, indicating that the recombinant enzymes are modified by N-linked complex-type carbohydrate moieties. In the human recombinant DNase I, activity was decreased by PNGase F-treatment, while that of the porcine DNase I remained unaffected. The thermal stability of the human enzyme was extremely susceptible to heat following PNGase F-treatment, as was the porcine enzyme to a lesser extent. This study suggests that N-linked complex-type carbohydrate moieties may contribute to the enzymatic activity and/or thermal stability of recombinant DNases I.
Fujihara
Junko
J
Department of Legal Medicine, Shimane University School of Medicine, Izumo 693-8501, Japan.
Hieda
Yoko
Y
Xue
Yuying
Y
Okui
Izumi
I
Kataoka
Kaori
K
Takeshita
Haruo
H
D000818:Animals; D019556:COS Cells; D005591:Chemical Fractionation / Q000379:methods*; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D037102:Lectins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013552:Swine
D037102:Lectins; D011994:Recombinant Proteins; D015253:Deoxyribonucleases, Type I Site-Specific