A novel host-vector system for direct selection of recombinant baculoviruses (bacmids) in Escherichia coli.
Site-specific transposition in Escherichia coli was used to introduce foreign genes into the Autographica californica nuclear polyhedrosis baculovirus genome. Using a temperature-sensitive donor plasmid and an E. coli host strain with an occupied Tn7 attachment site it was possible to select directly for 'bacmid' recombinants at 44 degrees C. A blue to white color screen provided further confirmation of insertion at the correct site in the baculovirus genome. After cloning the gene of interest into a donor plasmid, a single transformation and plating on selective medium resulted in homogeneous baculovirus DNA which could immediately be transfected into insect cells. The utility of the host-vector system for expression in insect cells was illustrated using three heterologous genes encoding beta-glucuronidase, human N-myristoyl transferase and murine preproguanylin. Using this approach, bacmid recombinants could be produced at a frequency of > or = 10(5) per micrograms input DNA. This system should not only greatly enhance the ability to obtain recombinant viruses for heterologous protein production, but should also be useful for protein engineering applications and expression cloning in insect cells.
Leusch
M S
MS
G.D. Searle and Co., St. Louis, MO 63198, USA.
Lee
S C
SC
Olins
P O
PO
D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D012641:Selection, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004279:DNA, Viral