Peter O. Olins

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.

Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing region-restricted substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, half-substituted variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the half-substituted variants was combined to yield fully substituted IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.