Product Details

pComb3X represents a novel iteration of the pComb vector series, building upon the foundational features of its predecessor, pComb3. Notable enhancements include improved stability and the integration of an asymmetric SfiI cassette, facilitating directional cloning of full Fabs, scFvs, peptides, and other proteins for phage display applications. The introduction of an amber stop codon allows for controlled expression of the pIII fusion protein, thus enabling the production of soluble protein without the need for additional subcloning steps. Furthermore, pComb3X incorporates separate cloning sites for heavy and light antibody chains, streamlining the construction of antibody libraries and enhancing downstream antibody identification processes.

Key Features of Our pComb3X Set

• Enhanced Stability: Compared to conventional phage display vectors, pComb3X offers improved stability, ensuring reliable and consistent performance over time.
• Streamlined Cloning Process: The incorporation of separate cloning sites for heavy and light antibody chains simplifies and accelerates the construction of antibody libraries, reducing experimental turnaround time.
• Precise Directional Cloning: The asymmetric SfiI cassette enables precise directional cloning of full Fabs, scFvs, peptides, and other proteins, enhancing cloning efficiency and accuracy.
• Controlled Protein Expression: The introduction of an amber stop codon allows for regulated expression of the pIII fusion protein, facilitating the production of soluble proteins without the need for additional manipulations.
• Versatile Application Range: pComb3X vectors are suitable for a wide range of high-throughput screening applications, including protein-protein interaction studies, selection of high-affinity binders, enzyme inhibitor screening, and ligand screening, among others.

pComb3X Set List

Application

Phage display panning is a powerful screening technique utilized in molecular biology research, wherein a library of proteins or peptides is genetically fused to a coat protein of a bacteriophage. This technique facilitates the identification of antibodies or proteins with enhanced affinity and specificity for target proteins. Phagemid pComb3, originating from the pBR322 plasmid, was among the pioneering phage display vectors used for generating and selecting antibody libraries in the 50-kDa Fab format—a monovalent surrogate for natural antibodies. Equipped with multiple restriction enzyme recognition sites, antibiotic resistance markers, and other genetic elements, pComb3 enabled a range of genetic manipulations in bacterial systems. The pComb3X series offers versatile applications:

• High-throughput protein-protein interactions screening: Employ the pComb3X series for efficient and comprehensive analysis of interactions between various proteins, facilitating the identification of novel protein complexes and functional networks.
• Specific binding protein selection: Utilize pComb3X vectors to isolate proteins with precise and potent binding capabilities, enabling the discovery of high-affinity binders for diverse applications in diagnostics, therapeutics, and biotechnology.
• Enzyme inhibitor screening: Utilize pComb3X constructs to screen for compounds capable of inhibiting enzyme activity, offering valuable insights into enzymatic mechanisms and potential therapeutic targets for drug development.
• Ligand screening: Employ pComb3X platforms to identify ligands with affinity for specific targets, facilitating drug discovery, molecular recognition studies, and elucidation of ligand-receptor interactions.
• Antibody library construction and screening: Utilize pComb3X vectors for the construction of diverse antibody libraries and subsequent screening for antibodies with desired specificity and affinity, enabling the development of novel diagnostics and therapeutics for various diseases.

Case Study

Case Study 1

Coxsackievirus A16 (CA16) is a primary etiological agent of hand, foot, and mouth disease (HFMD). Researchers utilized the pComb3X vector to express single-chain variable fragment (scFv) antibodies targeting CA16 infection. The vector, containing scFv genes, was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After five cycles of bio-panning, an effective scFv antibody with favorable binding activity to CA16-infected lysate proteins on ELISA plates was selected. This scFv clone demonstrated neutralizing capability against CA16 and cross-reacted with proteins in EV71-infected lysate. Additionally, polyclonal IgY antibodies also showed binding specificity against CA16-infected lysate proteins and significant neutralization activities. These findings suggest that both IgY and scFv antibodies may offer potential therapeutic options for treating CA16 infection, especially in vulnerable young children.

Figure 1. The pComb3X plasmid was utilized by researchers for cloning single-chain variable fragment (scFv) genes. It contained tags for the detection and purification of recombinant scFv antibodies, along with a g3p fragment enabling phage display of the scFv on viral particles. (Mwale PF, et al., 2021)

Case Study 2

The pComb3X vector was used by researchers to construct a large-sized naïve human antibody heavy chain domain library, to select virus neutralizing VH antibody domains. This protocol, which includes optimized steps for SARS-CoV-2 antigen expression, stable cell line construction, and library panning, led to the identification of VH ab8, a high-affinity neutralizing human VH antibody domain with potent prophylactic and therapeutic efficacy against SARS-CoV-2. For detailed execution and application of the protocol, readers are referred to Li et al.

Figure 2. pComb3X was used by researchers to allow translation to read through the amber codon and produce a full-length VH-gene III fusion protein in the amber codon suppressor strain TG1, facilitating extracellular expression studies. (Chen C, et al., 2021)

Case Study 3

Researchers utilized pComb3X for the establishment of a nanobody phage display library to target hemiustilaginoidin F-hapten coupled with bovine serum albumin (BSA). This approach facilitated heterologous antigen selection and nanobody screening, resulting in the identification of two nanobodies, Nb-B15 and Nb-C21. These nanobodies were then utilized to develop indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs) for quantifying total ustilaginoidins in rice samples. Nb-B15, selected for its superior properties, was further employed for detecting actual contaminated rice samples. The developed immunoassay proved to be a rapid and effective method for detecting total ustilaginoidins in contaminated rice samples, demonstrating the utility of pComb3X in nanobody-based assay development.

Figure 3. Using the pComb3X vector, a nanobody phage display library was constructed by researchers. After obtaining cDNA from blood samples, VHH genes were amplified and ligated with the vector. The library, estimated to have 1.84 × 10^9 colonies, exhibited good diversity, with a final titer of 1.18 × 10^13, achieved through helper phage rescue. (Wang W, et al., 2022)

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