Detection of host-derived contaminants in products of recombinant DNA technology in E. coli: a comparison of silver-staining and immunoblotting.
Contamination of medicines produced in E. coli by recombinant DNA methodology with host-cell proteins is considered a potential problem with this type of method. In this report techniques for the detection of trace quantities of host-cell proteins in SDS-gel electrophoretograms were examined. Detection of E. coli proteins by immunoblotting, using antisera raised in rabbits to lysates of E. coli, was compared with detection using the ultrasensitive silver stain. Silver staining detected a larger number of E. coli proteins in a one-dimensional electrophoresis system than did immunoblotting. Proteins that were markedly antigenic in the rabbit were detected at a greater sensitivity by the immunoblotting approach. Both techniques detected contaminant proteins in a preparation of methionyl human growth hormone produced in E. coli known to be contaminated with host-cell proteins. No contaminating proteins were seen by either technique in more rigorously purified preparations of growth hormone. A combination of these two approaches would provide useful evidence of purity of medicines produced by recombinant DNA technology, and is potentially applicable to a wide range of host-vector systems.
Gooding
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Bristow
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D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D004274:DNA, Recombinant; D004340:Drug Contamination; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D013006:Growth Hormone / Q000378:metabolism*; D007120:Immunochemistry; D011994:Recombinant Proteins; D012834:Silver; D013194:Staining and Labeling
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D012834:Silver; D013006:Growth Hormone