Protein sorting by high performance liquid chromatography. 2. Separation of isophosphorylates of recombinant human DNase I on a polyethylenimine column.
Anion exchange HPLC with a polyethylenimine (PEI) column separates recombinant human deoxyribonuclease I (rhDNase) glycoforms according to the extent and positions of phosphorylation of mannose residues in N-linked oligosaccharides. The separation provides a selectivity unavailable by anion exchange HPLC with other columns or by isoelectric focusing gel electrophoresis and can be used to quantify the phosphate content of preparations of rhDNase. Tryptic mapping of fractions collected from the column and treated with alkaline phosphatase was used to identify the sites of phosphorylation. Unnatural forms of rhDNase, bearing oligosaccharide structures at only one of the two sites of glycosylation, were prepared by cleaving the phosphate-containing high mannose and hybrid structures from the purified isophosphorylates fractionated on the PEI column. The separation of rhDNase isophosphorylates on the PEI column mimics the relative affinities for the mannose 6-phosphate receptor that traffics acid hydrolases to lysosomes and provides a useful example of protein sorting by biomimetic interaction chromatography.
Frenz
J
J
Department of Medicinal Analytical Chemistry, South San Francisco, California 94080.
Quan
C P
CP
Cacia
J
J
Democko
C
C
Bridenbaugh
R
R
McNerney
T
T
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D008358:Mannose / Q000032:analysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D010449:Peptide Mapping; D010766:Phosphorylation; D011094:Polyethyleneimine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D009844:Oligosaccharides; D011994:Recombinant Proteins; D011094:Polyethyleneimine; D003850:Deoxyribonuclease I; D008358:Mannose