Page 21 - Macromolecular Crystallography

10 MACROMOLECULAR CRYS TALLOGRAPHY

the production of biologically active eukaryotic the frequency of recombination during the produc-
proteins. tion of recombinant virus is low (Kitts and Possee,
Baculovirus expression is the most frequently 1993) and identification of recombinant plaques is
used method for expression in insect cells and difficult and time-consuming. Due to the obvious
employs Autographa californica nuclear polyhedrosis advantages of being able to produce large quan-
virus (AcNPV), a double stranded (ds) DNA virus tities of correctly folded, biologically active pro-
that infects arthropods. The baculovirus expres- tein, a number of improvements have been made
sion system utilizes features of the viral life cycle in order to make the production and propaga-
to introduce recombinant DNA coding the gene tion of recombinant virus more convenient and
of interest into insect cells (Miller, 1988; O’Reilly rapid. The Baculogold™(www.bdbiosciences.com)
et al., 1992). and the BacPak (www.clontech.com) systems uti-
The protein polyhedrin, which is produced in lize the method of linearization of Baculovirus DNA
large amounts during the very late phase of viral to increase the frequency of recombination. The
life cycle, acts to occlude virus particles and basis of this method is the introduction of rare
protects them from proteolysis during host cell restriction sites for the enzyme Bsu391 within the
lysis. However, polyhedrin is non-essential in the polyhedrin gene locus and the ORF1629 gene that is
viral life cycle (Smith et al., 1983). Another non- essential for viral replication (Possee and Howard,
essential protein expressed at high levels in the 1987; Kitts and Possee, 1993). Thus, linearization of
very late phase of the baculovirus life cycle is Baculovirus DNA with Bsu391 results in the exci-
p10 which is involved in polyhedra formation sion of the essential ORF1629 gene and renders this
(Williams, 1989; Vlak et al., 1988). Baculovirus DNA non-infective. Cotransfection of the linearized
expression systems take advantage of this since Baculovirus DNA with a transfer vector, containing
the protein of interest can be produced in large the missing sequence, restores infectivity. Moreover,
amounts by generating recombinant baculovirus since the gene of interest is within the ORF1629 locus
with the gene of interest replacing the poly- on the transfer vector, almost 100% recombinant fre-
hedrin or p10 genes with expression being driven quency can be achieved (Kitts and Possee, 1993).
by the polyhedrin or p10 promoter (Smith et It is important to remember that only the vectors
al., 1983). that contain the entire deleted region of the poly-
As the Baculovirus genome is too large (134 kb) hedrin gene can rescue the deletion by homologous
to be used as a cloning vehicle into which for- recombination.
eign genes can be inserted directly using stan- The most commonly used insect cell lines
dard molecular biology techniques, a transfer vec- Spodoptera frugiperda 9, 21 (Sf9, Sf21 respectively)
tor is used to insert the gene of interest into and HighFive (derived from Trichoplusia ni) (Geisse
the Baculovirus genome (Ayres et al., 1994). In et al., 1996) are commercially available from Invitro-
brief, the gene of interest is cloned into a (bacteri- gen and BD Biosciences. Healthy insect cells adhere
ally propagated) transfer vector flanked by viral- to the surface of the plate forming a monolayer
specific sequences. The transfer vector contain- and double every 18–24 h. Infected cells stop divid-
ing the gene of interest is then mixed with wild ing, become enlarged and uniformly round, have
type Baculovirus DNA and cotransfected into insect enlarged nuclei, and do not attach to the surface
cells. The gene is introduced into the Baculovirus of the plate. The possibility of cell growth in serum
genome, by homologous recombination, mediated free media with these cell lines also has the advan-
by the viral specific flanking sequences. Recom- tage of ease of purification of secreted proteins.
binant virus expressing the gene of interest can Insect cells can be grown both as monolayers and
be produced in this manner and the absence of in suspension. A culture is usually started as a
the polyhedrin gene allows identification of recom- monolayer (Protocol 1.5) and then transferred to
binant viral plaques since the viruses containing suspension into a spinner flask for large-scale pro-
polyhedrin have a different morphology. However, tein production. Healthy cells from a log-phase