A single-chain variable fragment (scFv) is a portion of a monoclonal antibody (MW ~26 kD), in which the variable immunoglobulin domains of the heavy (V
H) and light (V
L) chains are connected with a flexible linker into a single polypeptide chain (
Bird et al., 1988;
Huston et al., 1988). Thus, an scFv contains the entire antigen-binding region, and hence the specificity, of the parent antibody (
Bird et al., 1988;
Huston et al., 1988;
Sandhu, 1992;
Holliger and Hudson, 2005). The two variable domains are tethered together in either order (V
H–V
L or V
L–V
H), with a linker that is usually 10–25 amino acids in length, spanning the 35–40 Å distance from the C-terminus of one V domain to the N-terminus of the other (
Filpula et al., 1996;
Weisser and Hall, 2009). The length of this linker plays a crucial role for the oligomeric state of the soluble purified scFv (
Arndt et al., 1998;
Filpula et al., 1996), the most common linker being a 15mer (Gly
4Ser)
3 (
Huston et al., 1988;
Weisser and Hall, 2009).
Importantly, scFvs usually bind their cognate antigens with affinity similar to that of the parent antibody (
Bird et al., 1988;
Huston et al., 1988,
1996;
Skerra and Plückthun, 1988;
Weisser and Hall, 2009)–if the avidity effect of the bivalency of the latter is taken into account. This is due to the identical 3D arrangement of the variable domains. This feature, combined with the small size of scFvs, has made them attractive candidates for a wide range of applications, including therapeutics, medical imaging and diagnostics (
Begent et al., 1996). scFvs and scFv-based antibody fragments are currently in pre-clinical and clinical trials to treat human diseases ranging from heart disease to melanoma, and also for use in medical imaging (reviewed in
Holliger and Hudson, 2005). In addition, scFvs have shown promise in drug delivery systems and for the targeting of gene therapy vectors (
Glasgow et al., 2009;
Eisenstein, 2011).
The compact fold of scFvs, composed of two immunoglobulin domains each consisting of nine strands forming two tightly packed β-sheets stabilized by an intrachain disulfide bond, provides additional protein surfaces that can help to form a crystal lattice and thus promote crystallization of macromolecules (
Kovari et al., 1995;
Griffin and Lawson, 2011). Such an approach is particularly useful when dealing with proteins that are difficult to crystallize, such as heavily glycosylated proteins, or multidomain proteins with relatively flexible interdomain connections. Moreover, the compactness of scFvs makes them ideal candidates for structural studies on antigen–antibody binding, such as the characterization of the neutralization mechanism of certain monoclonal antibodies in viral infection (
Hwang et al., 2006;
Sui et al., 2009). These studies would be extremely difficult to perform using full-length bivalent antibody molecules, and are sometimes hindered by the flexibility of the elbow angle between variable and constant domains in Fab (fragment antigen-binding) fragments.
Various expression systems for scFvs have been reported, including
Escherichia coli, yeast, fungi, plants, insect and mammalian cells (
Bird et al., 1988;
Wu et al., 1993;
Jost et al., 1994;
Ridder et al., 1995;
Brocks et al., 1997); reviewed in
Verma et al. (1998) and
Weisser and Hall (2009). Numerous studies have been performed to evaluate quantity and quality of scFvs produced in different expression systems. The advantages and drawbacks of the most frequently used expression systems are summarized in Table . Importantly, in spite of numerous expression systems that have been explored for scFv expression, the expected effort required to produce large quantities of an scFv derived from a particular parent antibody strongly depends on the individual amino acid sequence (
Verma et al., 1998). Given that it is important to consistently obtain large quantities of pure scFv for structural analyses, as well as for diagnostic or therapeutic applications, we decided to further explore the use of the Drosophila Schneider 2 system for expression of scFvs. This system has been previously used to produce scFvs, but the reported expression levels varied by almost two orders of magnitude (0.2–20 mg/l) (
Mahiouz et al., 1998;
Reavy et al., 2000). High-yield expression of full-length monoclonal antibodies and Fab-fragments in S2 cells has also been reported (
Johansson et al., 2007a;
Backovic et al., 2010). The Drosophila S2 expression system is relatively easy to handle and is based upon logarithmically growing healthy cell lines that have been transfected to achieve stable expression of recombinant protein. This is in contrast to the production in insect cells using a recombinant baculovirus, which makes a lytic infection and induces considerable stress to the infected cell. Baculoviral-aided expression can therefore lead to saturation of the endoplasmic reticulum quality control system of the cell, especially when overexpressing proteins with complex, disulfide-stabilized folds. In such cases, baculovirus-induced cell lysis results in dumping of protein that has not gone through the ER quality control into the medium, resulting in a mixture of folded and partially folded protein in the supernatant.
| Table I.Advantages and disadvantages of different expression systems for scFvs |
We undertook to establish an expression system for scFvs that would allow easy cloning of the variable domains of heavy and light chain and production of large amounts of correctly processed and secreted scFvs using Drosophila S2 cells. We created a panel of scFvs from both human and murine parent antibodies directed against various antigens. In this paper we discuss in particular the production of five of the recombinant proteins-scFv 3H5, derived from a murine antibody to Npro of bovine viral diarrhea virus (BVDV); scFv 8B9 and scFv 6A5, both derived from murine antibodies to glycoprotein E2 of BVDV; and scFvs 1:7 and A8, both derived from human antibodies to hepatitis C virus glycoprotein E2 (
Allander et al., 2000).
