Analysis of recombinant human interleukin-11 fusion protein derived from Escherichia coli lysate by combined size-exclusion and reversed-phase liquid chromatography.
A two-dimensional size-exclusion-reversed-phase high-performance liquid chromatographic assay has been developed for the quantitation of recombinant human interleukin-11 fusion protein (rhIL-11 FP) expressed in E. coli cells. The sample preparation procedure included the optimization of lysis buffer components to achieve maximum rhIL-11 FP recovery through the disruption of associations between rhIL-11 FP and E. coli components. The E. coli cells were dialyzed into lysis buffer and lysed by a French Press prior to two-dimensional chromatographic analysis. A size-exclusion column was used first to remove high- and low-molecular-mass E. coli components. Then reversed-phase chromatography was used to separate and quantify the rhIL-11 FP. The assay was linear over the range of 0.0294 to 0.235 mg/ml. The limit of quantitation, 0.0294 mg/ml, was based on % normalized residuals and precision criteria not exceeding 10%. The reproducibility of the assay for lysate samples was good on a daily (% R.S.D. = 1.0; n = 5) and a day-to-day reproducibility was good (% R.S.D. = 2.2; n = 9). Selectivity and chromatographic peak identification were based upon gel electrophoresis and N-terminal sequencing of the rhIL-11 FP peak collected from the reversed-phase column.
Amari
J V
JV
Genetics Institute, Andover, MA 01810, USA.
Mazsaroff
I
I
D002021:Buffers; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D006801:Humans; D017370:Interleukin-11 / Q000032:analysis* / Q000096:biosynthesis; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012015:Reference Standards; D012965:Sodium Chloride / Q000737:chemistry
D002021:Buffers; D017370:Interleukin-11; D011994:Recombinant Proteins; D012965:Sodium Chloride