Appl Microbiol Biotechnol (2010) 86:1067–1075 1071 Fig. 1 Construction of HTNV GPs targeting scFv3G1. a PCR for the construction of scFv antibody-encoding gene. The genes encoding the variable heavy chain (VH) and variable light chain (VL) fragments were amplified and assembled via a short nucleotide linker in a VH-linker-VL format. The amplified products were resolved in 1.5% agarose gel and stained with ethidium bromide. DNA markers are given in base pairs indicated at the left. b Nucleotide and deduced amino acid sequences of scFv3G1. The linker fragment (Gly4Ser)3 is shown in italics. The complementarity- determining regions (CDRs) of the VH (H-CDR) and VL (L-CDR) domains are underlined, and the sequences were identical to those reported in the Kabat database. The four conserved Cys are indicated by stars The SDS-PAGE of crude cytoplasmic preparation and purified recombinant protein showed a prominent band at the predicted size for scFv3G1 fusion protein molecular weight of about 46 kDa (Fig. 2a), which was detected in all fractions from induced bacteria culture. This His-tagged protein was further examined by Western blot using anti- His-Tag antibody (Fig. 2b). These results showed that this recombinant scFv3G1 protein was released into cytoplasm as a soluble protein, and no degradation products were revealed, suggesting the high solubility and stability of the recombinant protein. The average concentration of affinity- purified protein was 1 mg/mL. In total, about 8 mg of soluble recombinant protein scFv3G1 with more than 90% purity was obtained per 1 L of E. coli culture. scFv3G1 specifically recognizes target antigen and retains the antigen-binding activity In order to investigate the antigen-specific binding of the recombinant scFv3G1, a solid-phase assay was performed using coated plates with HTNV GP as a target antigen. Within the rang tested, increased concentrations of scFv3G1 corresponded to enhanced colorimetric signals, while no significant background was detected in wells coated with the control HTNV NP antigen (Fig. 3a). Moreover, the scFv3G1 showed clear reactivity similar to the parental MAb3G1 (Fig. 3b). The observed difference in binding presumably shows reduced avidity of scFv, based Fig. 2 Expression and purification analysis. Escherichia coli BL21 (DE3) was transformed with the pET32a-scFv3G1 construct. After induction, cytoplasmic proteins were extracted following sonication. Cytoplasmic fraction was purified by immobilized metal affinity chromatography. Bacterial cytoplasmic extract and purified recombi- nant scFv3G1 antibody were subjected to 15% SDS-PAGE, stained with Coomassie brilliant blue (a) or transferred to PVDF membrane for Western blot analysis using anti-His-Tag antibody (b). c Western blot analysis of immunoreactivity of scFv3G1. HTNV GP or unrelated HTNV NP was transferred to PVDF membrane, incubated with scFv3G1, and followed by incubation with HRP-conjugated anti-His- Tag antibody. Molecular weight markers are given in kilodaltons indicated on the left