Capillary electrophoresis for purity estimation and in-process testing of recombinant GB virus-C proteins.
Protein purity estimation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with scanning densitometry is a critical component in the manufacture of recombinant proteins for commercial diagnostic assays. However, the procedure is time consuming and often difficult to reproduce because commercial dyes that are used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes the use of a rapid and dye-independent SDS polymer-filled capillary gel electrophoresis (CE-SDS) method to estimate protein purity. The CE-SDS method was used for in-process and final purity testing of GB virus-C (GBV-C) fusion proteins produced in E. coli, and was directly compared with the conventional SDS-PAGE method using purified Coomassie blue dye to reduce protein staining anomalies. The CE-SDS method may serve as an alternative or replacement method to the lengthy and tedious SDS-PAGE method. This study also demonstrates that the observed molecular weight of the fusion protein, determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), provides higher accuracy than values estimated by either CE-SDS or SDS-PAGE methods.
Kundu
S
S
Diagnostics Division, Abbott Laboratories, North Chicago, IL 60064, USA.
Fenters
C
C
Lopez
M
M
Varma
A
A
Brackett
J
J
Kuemmerle
S
S
Hunt
J C
JC
D003001:Cloning, Molecular; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli; D018067:Flaviviridae; D016679:Genome, Viral; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000592:standards; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000592:standards*; D015203:Reproducibility of Results; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000302:isolation & purification; D014764:Viral Proteins / Q000302:isolation & purification* / Q000592:standards; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases