title: Determination of recombinant human proinsulin fusion protein produced in Escherichia coli using oxidative sulfitolysis and two-dimensional HPLC.
abstract: A method for the sample preparation and determination of a human proinsulin fusion protein (ChPI) expressed in recombinant Escherichia coli samples is described. The method is applicable to samples containing whole cells or isolated inclusion bodies. The procedure involves the rapid sulfitolysis of samples in 7 M guanidine hydrochloride and analysis with a column-switch method using size exclusion and weak anion exchange HPLC. The response of the method was linear for ChPI-S-sulfonate concentrations up to 4.4 mg/mL. Recovery of standard added to samples was greater than 95% in all cases. The specificity of the method was demonstrated by the analysis of E. coli cells containing a negative control plasmid. The reproducibility of the method was good on a daily (% RSD = 1.618; n = 18) and a day-to-day (% RSD = 3.346; n = 26 days) basis. The general applicability of this approach was suggested by quantitating recombinant trypsinogen (methionyltrypsinogen expressed in E. coli), as the S-sulfonate, using size exclusion and cation exchange HPLC.
authors.affiliation: Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285.
authors.forename: J S
authors.initials: JS
authors.lastname: Patrick
authors.forename: A L
authors.initials: AL
authors.lastname: Lagu
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D011384:Proinsulin / Q000032:analysis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis*
chemical_list: D006146:Guanidines; D011993:Recombinant Fusion Proteins; D011384:Proinsulin; D019791:Guanidine