PREPARATION OF ASIALOOROSOMUCOID POLYLYSINE CONJUGATES

Citation
Td. Mckee et al., PREPARATION OF ASIALOOROSOMUCOID POLYLYSINE CONJUGATES, Bioconjugate chemistry, 5(4), 1994, pp. 306-311
Citations number
32
Categorie Soggetti
Biology,Chemistry
Journal title
ISSN journal
10431802
Volume
5
Issue
4
Year of publication
1994
Pages
306 - 311
Database
ISI
SICI code
1043-1802(1994)5:4<306:POAPC>2.0.ZU;2-Z
Abstract
Asialoorosomucoid-polylysine (ASOR-PL) conjugates have been recently d eveloped as carriers of electrostatically bound DNA for targeted deliv ery to the hepatic asialoglycoprotein receptor (ASGPr) for gene therap y. Using acid-urea gel electrophoresis we have found that previously r eported procedures for the fractionation of ASOR-PL conjugates do not efficiently remove noncovalently bound polylysine (PL) from ASOR-PL. D NA complexes prepared with these conjugates have low solubilities, whi ch limits their usefulness for subsequent experimentation, particularl y in vivo. For ASOR-PL made by carbodiimide-mediated crosslinking with 5-kDa PL, dialysis against 1 M guanidine hydrochloride is effective t o remove the low molecular weight unbound PL. Dialysis is not feasible when using higher molecular weight PLs, but preparative elution acid- urea gel electrophoresis was used to isolate crude ASOR-PL fractions f ree of unbound PL. ASOR-PL freed of PL by dialysis or electrophoresis was further fractionated by cation-exchange HPLC on carboxymethyl-func tionalized columns eluted with a mixed pH-salt gradient. Early-eluting ASOR-PL fractions isolated by a combination of preparative elution ac id-urea gel electrophoresis and cation-exchange HPLC were found to be preferred for the formation of soluble DNA complexes.