Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization.

A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.

Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions.

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.