A platform-agnostic, function first-based antibody discovery strategy using plasmid-free mammalian expression of antibodies.

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery. In this study, we describe a process to identify and rapidly re-format and express antibodies for functional characterization. We used two different approaches to isolate antibodies to five different targets: 1) flow cytometry to identify antigen-specific single B cells from the spleen of immunized human immunoglobulin transgenic mice; and 2) panning of phage libraries. PCR amplification allowed recovery of paired VH and VL sequences from 79% to 96% of antigen-specific B cells. All cognate VH and VL transcripts were formatted into transcription and translation compatible linear DNA expression cassettes (LEC) encoding whole IgG or Fab. Between 92% and 100% of paired VH and VL transcripts could be converted to LECs, and nearly 100% of them expressed as antibodies when transfected into Expi293F cells. The concentration of IgG in the cell culture supernatants ranged from 0.05 µg/ml to 145.8 µg/ml (mean = 18.4 µg/ml). Antigen-specific binding was displayed by 78-100% of antibodies. High throughput functional screening allowed the rapid identification of several functional antibodies. In summary, we describe a plasmid-free system for cloning and expressing antibodies isolated by different approaches, in any format of choice for deep functional screening that can be applied in any research setting during antibody discovery.

Cloning and Detection of Aptamer-Ribozyme Conjugations.

RNA aptamers can be used to target proteins or nucleic acids for therapeutic purposes and are candidates for RNA-mediated gene therapy. Like other small therapeutic RNAs, they can be expressed in cells from DNA templates that include a cellular promoter upstream of the RNA coding sequence. Secondary structures flanking aptamers can be used to enhance the activity or stability of these molecules. Notably, flanking self-cleaving ribozymes to remove extraneous nucleotides included during transcription as well as flanking hairpins to improve RNA stability have been used to increase the effect of therapeutic aptamers. Here we describe the cloning procedure of aptamers containing different flanking secondary structures and methods to compare their expression levels by a northern blot protocol optimized for the detection of small RNA molecules.

Natural Genetic Transformation: A Direct Route to Easy Insertion of Chimeric Genes into the Pneumococcal Chromosome.

The ability of Streptococcus pneumoniae (the pneumococcus) to transform is particularly convenient for genome engineering. Several protocols relying on sequential positive and negative selection strategies have been described to create directed markerless modifications, including deletions, insertions, or point mutations. Transformation with DNA fragments carrying long flanking homology sequences is also used to generate mutations without selection but it requires high transformability. Here, we present an optimized version of this method. As an example, we construct a strain harboring a translational fusion ftsZ-mTurquoise at the ftsZ locus. We provide instructions to produce a linear DNA fragment containing the chimeric construction and give details of the conditions to obtain optimal pneumococcal transformation efficiencies.

Construction and application of EV71 ScFv high-throughput cassette expression system / 中华实验和临床病毒学杂志

Objective@#To construct a method to express ScFv antibody from PCR products, and use it in phage display for high-throughput ScFv expression.@*Methods@#Cytomegalovirus (CMV) promotor, ScFv and BGH-Poly A gene fragments were amplified by PCR. Overlapping PCR was used to form a tandemly linear cassette gene. By transiently transfected into 293T cells, ScFv antibodies expression of cassette gene were tested by Western blot, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody (IFA). Ninety-six clones of antibody genes in phage library were selected and expressed by cassette expression system. The expression level was evaluated and analyzed.@*Results@#Three fragments were obtained and a cassette expression system formed. Cassette expression system worked successfully in 293T cells, as a Mr.38×103 brand could observed in Western blot assay. The expressed antibody could specifically bind to its antigen by result of ELISA and IFA. This cassette expression system could also be used in phage display for high-throughput panning.@*Conclusions@#The cassette expression system was constructed successfully and high-throughput antibody expression has been achieved.

Using overlap-extension PCR technique to fusing genes for constructing recombinant plasmids.

Overlap-extension PCR is a method for splice of gene segments to produce focused fragments for constructing recombinant plasmid, but its complexity limits its application. To simplify the protocol and to improve the effectiveness, we employed gradient temperatures to replace the single annealing temperature in the thermo-cycling program, and optimize the templates ratio. The concentration of each fragment was adjusted to 10 ng µl-1 . Fragment concentration ratio was the inverse of the fragment size ratio. The products of fused segments were 2000-5000 bp in length using the revised one-step method. This method splices effective two or more fragments to fused gene and produce recombinant plasmid.

A Guide to Using STITCHER for Overlapping Assembly PCR Applications.

Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping assembly PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online.

[Construction and identification of BKCaαsubunit expression plas-mid with double labeling of Flag and GFP].

OBJECTIVE: This study aimed to construct a large conductance calcium activated potassium channel α (BKCa) subunit plasmid with two tags by the overlapping PCR technique to set up a steady base for future ion channel study. METHODS: Based on the existing coding BKCa channel α subunit expression plasmid pcDNA3.1-hSlo, we constructed a double-tag expression plasmid, namely, pcDNA3.1-Flag-hSlo-GFP (Flag-hSlo-GFP). RESULTS: Flag tag was inserted into the S1-S2 extracellular loop of BKCa channel α subunit, and GFP tag was connected to the C-terminus of BKCa channel α subunit. Sequence of the constructed plasmid was confirmed successful. CONCLUSIONS: The expression plasmid Flag-hSlo-GFP was constructed successfully with overlapping PCR. Overlapping PCR is a valuable method for amplifying long size genes.

STITCHER: A web resource for high-throughput design of primers for overlapping PCR applications.

Overlapping PCR is routinely used in a wide number of molecular applications. These include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping by traditional PCR techniques and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online (http://ohalloranlab.net/STITCHER.html). STITCHER can handle both single sequence and multi-sequence input, and specific features facilitate numerous other PCR applications, including assembly PCR, adapter PCR, and primer walking. Field PCR, and in particular, LAMP, offers promise as an on site tool for pathogen detection in underdeveloped areas, and STITCHER includes off-target detection features for pathogens commonly targeted using LAMP technology.