Technology
In Vivo Production of Recombinant Proteins
The full power of the Baculovirus Expression Vector System
Chesapeake PERL’s production system is a proprietary in vivo implementation of the well-established Baculovirus Expression Vector System (BEVS). Building on the favorable characteristics of the BEVS noted below, C-PERL’s larval system offers significant advantages over other systems for the production of high levels of functional, biologically active recombinant proteins, including:
Tissue Heterogeneity. Proteins express differently in different tissues. In contrast to the homogeneity of cultured cells, the tissue heterogeneity of in vivo production can lead to both higher levels of expression and more native modification and processing. This is especially true for proteins that are not secreted, such as membrane-bound or membrane-associated proteins, or for multimeric proteins. In addition, host protein synthesis remains active in some cell types, providing enzymes, lipoproteins, chaperones and other elements that can enhance protein expression. Examples of improved processing include propeptide cleavage, C-terminal cleavage, dimerization, complex formation, association with lipids and formation of heterodimers.
C-PERL’s system is highly automated and produces uniformly inoculated insect larvae for highly reproducible results. Unlike other production systems, the larval system scales linearly without re-optimization. Whether the need for biomass from which to purify a protein is ten grams or hundreds of kilograms per week, production of larvae and the recombinant proteins they contain is predictable, with none of the mixing, aeration or flow issues confronted when scaling cells in culture. The larval system also eliminates the multiple passage in cell culture. We routinely produce and store sufficient POV from the second passage to produce very large quantities of recombinant protein-containing larvae.
Small quantities of inoculated biomass from a baculovirus construct, for example for small-scale protein production or candidate screening, can be prepared in four days. Larger quantities are available in less than two weeks from the initial insect inoculation.
The use of larvae for the production of recombinant proteins was pioneered with the silkworm, Bombyx mori, for the production of interferon (Maedaet al., 1985) and mouse interleukin-3 (Miyajima et al., 1987). C-PERL substituted Trichoplusia ni, the cabbage looper, for the silkworm because it is an excellent host for AcMNPV (Autographa californica multiple nucleopolyhedrosis virus), the most highly developed baculovirus vector, and because its life cycle lends itself to the automation of inoculation, incubation and harvesting.
The value of C-PERL’s technology comes from its application to the production of specific proteins for our partners and customers. You will find case studies [Case Study], a list of the proteins we have made [Proteins Produced], and a list of proteins available for sale from our catalog [Products]. Contact us to discuss how you can benefit from C-PERL’s unique recombinant protein production capabilities.
BEVS has been used for more than twenty five years for the production of recombinant proteins from insect cells. Most commonly, an Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) engineered to contain a target gene is used to infect Sf9 cells developed from Spodoptera frugiperda, the fall armyworm, or Hi5 cells developed from Trichoplusia ni, the cabbage looper. Expression levels of heterologous protein as high as 30-50% of cellular protein have been reported, but very high expression levels can overwhelm the cellular processes responsible for post-translational modification, folding and transport. In some cases this biological bottleneck can be overcome through the expression of a protein in a more appropriate larval tissue.
Protein Modification and Processing. Under optimal conditions the recombinant protein is modified, disulfide bonds are formed and the protein folds properly. Observed post-translational modifications include N- and O-linked glycosylation, phosphorylation, acylation, amidation, carboxymethylation, isoprenylation, signal peptide cleavage and proteolytic cleavage, with the modification sites corresponding to those of the protein in its native environment. In some cases larvae modify and process proteins to more closely resemble native proteins than cultured cells do.
Gene Capacity. There is no known limit to the size of the gene insert and thus multiple genes can be expressed simultaneously, leading to the production of heteromultimeric proteins or proper processing of a protein that requires a species- or tissue-specific modifying enzyme.
Baculoviruses have a restricted host range, limited to specific invertebrate species, and are non-infectious to vertebrates, while mammalian pathogens, including viruses and prions, cannot propagate in insect cells. In addition, C-PERL uses a proprietary orally infectious form of the recombinant virus that is unstable in the environment, enhancing the safety of the platform.
The culture medium in which the insect cells are grown is significantly less expensive than that used for mammalian cells. C-PERL’s larval system is based on an inexpensive, pasteurized wheat germ/agar medium, which further reduces cost.