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 b aculovirus genome. Using a temperature-sensitive donor plasmid and an E. coli host strain with an occupied Tn7 attachment site it was possib le to select directly for 'bacmid' recombinants at 44 degrees C. A blu e 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 i mmediately be transfected into insect cells. The utility of the host-v ector system for expression in insect cells was illustrated using thre e heterologous genes encoding beta-glucuronidase, human N-myristoyl tr ansferase and murine preproguanylin. Using this approach, bacmid recom binants could be produced at a frequency of greater than or equal to 1 0(5) per mu g input DNA. This system should not only greatly enhance t he ability to obtain recombinant viruses for heterologous protein prod uction, but should also be useful for protein engineering applications and expression cloning in insect cells.