杆状病毒表达系统(The Baculovirus Expression System)

Baculovirus Facility in Cambridge

The Baculovirus Expression System

The Baculovirus Expression Vector System (BEVS) has been widely used in research and scientific industrial communities for the production of high levels (up to 1000mg/mL) of properly post-translationally modified (folding, disulfide bond formation, oligomerization, glycosylation, acylation, proteolytic cleavage), biologically active and functional recombinant proteins. The Baculovirus Expression Vector System is based on the introduction of a foreign gene into nonessential for viral replication genome regio n via of homologous recombination with a transfer vector containing target gene. The resulting recombinant Baculovirus lacks one of nonessential gene (polh, v-cath, chiA etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae. Baculovirus Facility in Cambridge offers services on protein expression and production - from recombinant baculovirus production to large scale protein expression.

Several features make the Baculovirus Expression Vector System attractive for researchers:

- High levels of heterologous gene expression are often achieved compared to other eukaryotic expression systems, particularly for intracellular proteins. In many cases, the recombinant proteins are soluble, post-translationally modified and easily recovered from infected cells late in infection when host protein synthesis is diminished.

-The cell lines used for AcMNPV propagation grow well in suspension cultures, permitting the production of recombinant proteins in large-scale bioreactors.

- Expression of hetero-oligomeric protein complexes can be achieved by simultaneously infecting cells with two or more viruses or by infecting cells with recombinant viruses containing two or more expression cassettes.

- Baculoviruses have a restricted host range, limited to specific invertebrate species. They are safer to work with than most mammalian viruses since they are noninfectious to vertebrates.

What is baculovirus?

Baculoviruses are rod-shaped (baculum in Latin means 'stick') dsDNA viruses found mainly in insects. The most common baculovirus used for expression studies is Autographa californica multiple nuclear polyhedrosis virus (Ac MNPV), which relies on the lepidopteran species Spodoptera frugiperda and Trichoplusia ni as host insects. Ac MNPV particles surround themselves with a protective matrix consisting of the protein polyhedrin, which permits survival in the environment and efficient spread to new hosts.

Under the control of the extremely strong promoter pPolh, polyhedrin is expressed at extremely high levels (up to 50% of all cellular protein) at the end of the baculovirus life cycle. The baculovirus expression system makes use of the fact that in cell culture a polyhedrin coat is not essential for virus propagation and thus heterologous proteins can be expressed under the control of the pPolh promoter (see O'Reilly et al. (1997) for further reading).

Bac-to-Bac procedure

The Bac-to-Bac baculovirus system (Invitrogen) provides a rapid way for the generation of recombinant baculoviruses. It is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid). Bacmid DNA can be propagated in E. coli DH10Bac as a large plasmid that confers resistance to kanamycin. The bacmid complements a lacZ deletion present on the chromosome to form colonies that are blue (Lac-) in the presence of a chromogenic substrate and the inducer IPTG. Transposition of the gene of interest from the pFastBac1 donor plasmid into the bacmid disrupts the lacZ sequence causing recombinant bacmids to appear as white colonies. Transfection of the recombinant bacmid DNA into insect cells leads to the generation of recombinant baculovirus particles that can be amplified in successive rounds of infection and eventually used for protein expression studies (see Invitrogen manual for further details)