Exploiting features of adenovirus replication to support mammalian kinase production.
Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.
Cotten
Matt
M
Axxima Pharmaceuticals AG, Max-Lebsche-Platz 32, 81377 München, Germany. matt.cotten@axxima.com
Stegmueller
Kerstin
K
Eickhoff
Jan
J
Hanke
Miriam
M
Herzberger
Katrin
K
Herget
Thomas
T
Choidas
Axel
A
Daub
Henrik
H
Godl
Klaus
K
D000256:Adenoviridae / Q000187:drug effects / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002846:Chromatography, Affinity; D004340:Drug Contamination; D004789:Enzyme Activation; D005818:Genetic Engineering; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D008322:Mammals / Q000821:virology; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011494:Protein Kinases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays; D014779:Virus Replication
D007364:Intercalating Agents; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D011494:Protein Kinases