Specific N-terminal amino acids potentiate the periplasmic expression of single-chain variable fragments in Escherichia coli.

Single-chain variable fragments (ScFvs) are important in therapy, diagnosis and research because of their elevated antigen affinity and low immunogenicity. At present, high-yield scFv expression in Escherichia coli is limited by insoluble aggregation in the reducing environment of the cytoplasm or low yields in the periplasm. Here we achieved increased expression of scFvs in the periplasm by inserting optimal amino acids between the signal peptide and scFv. We constructed an expression library with three random amino acids at the scFv N-terminus, screened this library with a single-step colony assay and identified the specific sequences that boosted periplasmic expression of scFvs.

High-yield expression of periplasmic single-chain variable fragments by solid Escherichia coli cultures.

High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in Escherichia coli using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in E. coli cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated E. coli. This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.

Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins.

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and-most importantly-it enables high-throughput procedures.

Potentiating Antigen-Specific Antibody Production with Peptides Obtained from In Silico Screening for High-Affinity against MHC-II.

Monoclonal antibodies with high affinity and specificity are essential for research and clinical purposes, yet remain difficult to produce. Agretope peptides that can potentiate antigen-specific antibody production have been reported recently. Here, we screened in silico for peptides with higher affinity against the agretope binding pocket in the MHC-II. The screening was based on the 3D crystal structure of a complex between MHC-II and a 14-mer peptide consisting of ovalbumin residues 323-339. Using this 14-mer peptide as template, we constructed a library of candidate peptides and screened for those that bound tightly to MHC-II. Peptide sequences that exhibited a higher binding affinity than the original ovalbumin peptide were identified. The peptide with the highest binding affinity was synthesized and its ability to boost antigen-specific antibody production in vivo and in vitro was assessed. In both cases, antigen-specific IgG antibody production was potentiated. Monoclonal antibodies were established by in vitro immunization using this peptide as immunostimulant, confirming the usefulness of such screened peptides for monoclonal antibody production.

Single-step colony assay with autoinduction of scFv expression for the screening of antibody libraries.

We present a simple colony assay method for screening antibody libraries based on autoinduction of antibody fragment expression. This protocol eliminates the need for colony size monitoring and a separate induction step for single-chain Fv (scFv) antibody fragment expression. Here, scFvs are expressed in an automatic and timely fashion during the assay, resulting in high yields of positive clones and substantial time savings. The method was used successfully to establish monoclonal scFvs with high affinity and specificity against human IgG.

Single-step colony assay for screening antibody libraries.

We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×103 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries.

Enzymatic Assembly for scFv Library Construction.

Recombinant monoclonal antibodies can be established by displaying single-chain variable fragment (scFv) antibody libraries on phages and then biopanning against the target. For constructing superior scFv libraries, antibody light-chain variable region (VL) and heavy-chain variable region (VH) fragments must be assembled into scFvs without loss of diversity. A high-quality scFv library is a prerequisite for obtaining strong binders from the scFv library. However, the technical challenges associated with the construction of a diverse library have been the bottleneck in the establishment of recombinant antibodies through biopanning. Here, we describe a simple and efficient method for assembling VL and VH fragments through the concerted action of λ-exonuclease and Bst DNA polymerase. We successfully used this method to construct a diverse chicken scFv library.

Potentiation of antigen-specific antibody production by peptides derived from Ag85B of Mycobacterium tuberculosis.

To generate high-titer monoclonal antibodies, strong immuno-stimulation must be used for eliciting an intense cellular immune response. Here, we report that antigen-specific antibody production was potentiated by Peptide-25 derived from Ag85B of Mycobacterium tuberculosis, and that the production of antigen-specific IgG1 in particular was markedly potentiated; specifically, this occurred because the use of Peptide-25 resulted in an increase in the number of antigen-specific antibody-producing cells. We studied the activation of T cells by the peptide by examining gene expression. The observed expression pattern of GATA-3 and T-bet suggests that the peptide modulates the Th1/Th2 balance during immunization. This potentiation, which was remarkably high in BALB/c mice, could be applied in the immunization performed for monoclonal antibody production in vivo and in vitro.

Construction of an scFv library by enzymatic assembly of V(L) and V(H) genes.

The single-chain Fv fragment (scFv) is the most frequently used form of recombinant antibody. It is possible to establish clones specific to a certain target by displaying the scFv library on phages followed by biopanning against the target. For the construction of superior scFv libraries, the light-chain variable region (VL) and the heavy-chain variable region (VH) fragments should be assembled into the scFv without loss of diversity. We have provided an efficient method for constructing scFvs by enzymatic assembly of the VL and VH domains using the concerted action of λ-exonuclease and Bst DNA polymerase. First, we amplified the chicken VL and VH fragments using a phosphorylated primer with a 21-nucleotide overlap in the linker region. Then we recessed the overlapping parts of the VL and VH fragments with λ-exonuclease, which yielded single-stranded overhangs that specifically annealed between the VL and VH fragments; the complete double-stranded scFv was formed using Bst DNA polymerase. Complete scFvs were obtained using this method, whereby a library of scFvs was constructed from the immune library of chicken IgG. The diversity of this scFv library was analyzed by DNA fingerprinting method. The scFvs specific to the antigen could be isolated from this library after 5 rounds of panning.

A method for inducing antigen-specific IgG production by in vitro immunization.

In vitro immunization (IVI) possesses a number of advantages over conventional immunization. However, the number of positive clones derived from IVI is limited, and the affinity of the antibodies from derived clones is relatively low. Moreover, the majority of immunoglobulins produced in culture are IgMs instead of IgGs, which limits the application. Here, we report an improved protocol for IVI using mouse spleen cells. This protocol consists of multiple cycles of repeated antigen stimulation followed by cell expansion, which increases the frequency of plasma cells that produce antigen-specific IgG antibodies. The culture conditions, including the cell density, the type of stimulants, and the initial cell preparation, were found to be important for inducing the IgG response. In addition, an analysis of the genes and cytokines expressed during the IVI showed that the antigen-specific B cells were specifically activated via CD4-positive helper T cells. As evidence for this concept, our IVI protocol enabled us to establish an IgG antibody against keyhole limpet hemocyanin with a dissociation constant in the order of 10(-7)M.

Cell activation by CpG ODN leads to improved electrofusion in hybridoma production.

Hybridoma formation is an indispensable step in the production of monoclonal antibodies. Obtaining highly efficient fusion of an antibody-producing cell to the myeloma cell to form the hybridoma is an important step in this process. The electrofusion method is superior to chemical fusion methods such as the polyethylene glycol (PEG) method due to its high fusion efficiency. However, this method requires cell activation prior to electrofusion, a process that is time-consuming and tends to cause cell death. In this study, we achieved much higher fusion efficiency by stimulating B cells with CpG oligodeoxynucleotide (CpG ODN) over shorter periods. Splenocytes were isolated from immunized mice and cultured in the presence of a CpG ODN for 1 or 2 days. This CpG ODN stimulation evokes about one order of magnitude higher fusion efficiency than other stimulators. CpG ODN stimulation not only increases the fusion efficiency but also the number of antibody-producing cells. This leads to a substantial increase in the number of positive clones obtained. This highly efficient fusion method was used to produce a functional antibody against Gaussia luciferase. This method was found to produce greater numbers of hybridomas and to enable direct screening for antibodies with functional characteristics such as inhibition of the luminescence activity of an antigen. We were able to establish a functional antibody against Gaussia luciferase after a single fusion experiment using our electrofusion method.

Salt bridges in prion proteins are necessary for high-affinity binding to the monoclonal antibody T2.

We studied the role of the 2 salt bridges (Asp143-Arg147 and Asp146-Arg150) in helix 1 of mouse prion protein (PrP) on the formation of the complex between PrP and the monoclonal antibody T2. We introduced 6 charge-changing mutations to the amino acid residues associated with the salt bridges. Analysis of the circular dichroism spectra of the mutant PrPs showed that the salt bridge mutations did not change the secondary structures. We analyzed the kinetics of the association and dissociation of the PrPs with the T2 antibody. The results showed that the association kinetics were not significantly different among the variants except Arg150Lys, while the dissociation rate of the neutralized-charge variants was 2 orders of magnitude higher than that of the wild type. These results indicate that salt bridges make the interaction of PrP with T2 tighter by slowing down dissociation.

Bioluminescence assay for detecting cell surface membrane protein expression.

We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.

Characterization of discontinuous epitope of prion protein recognized by the monoclonal antibody T2.

The anti-prion protein (PrP) monoclonal antibody T2 has previously been prepared using PrP-knockout mice immunized with mouse recombinant PrP residues 121-231, however its interaction mechanism to PrP antigen has not been cleared. Here we identified and characterized the epitope of T2 antibody. The competitive ELISA with 20-mer synthetic peptides derived from PrP121-231 showed that T2 antibody had no affinity for these peptides. The analysis with deletion mutants of PrP revealed that 10 amino acids in the N terminus and 66 amino acids in the C terminus of PrP121-231 were necessary for reactivity with T2. Two far regions are necessary for complete affinity of the T2 antibody for PrP; either region alone is not sufficient to retain the affinity. The epitope recognized by T2 antibody is discontinuous and conformational. We examined the effect of disulfide bond and salt bridges. Alkylation of cysteine residues in C terminus of PrP121-231, which breaks a disulfide bond and disrupts the structure, had diminished the reactivity. Mutations induced in the PrP121-231 to break the disulfide bond or salt bridges, markedly had reduced the reactivity with T2 antibody. It suggests that T2 antibody recognized the structure maintained by the disulfide bond and salt bridges.