Creative Biolabs has been a long-term expert and market leader in the field of phage display and antibody engineering. Through our unparalleled phage display platform, Creative Biolabs offers integrated service of cDNA library construction to our customers over the globe.

Background

Complementary DNA (cDNA) is the reverse transcript of mRNA. As lacking the large non-coding introns compared to genomic DNA, eukaryotic cDNA libraries represent the coding sequence of all temporally transcribed genes in a certain cell type. Aided by phage display technology, the entire cDNA products can be expressed on the phage surface by fusion proteins, thus allowing identification of a large panel of protein candidates as well as isolating specific binders towards various targets. Over the past decades, phage display cDNA libraries have been widely utilized for antigen/antibody discovery, vaccine design, clinical therapies and diagnosis, etc.

Conventional phage display technique meets obstacles in constructing complex cDNA libraries, such as low concentration of target mRNA, frequent in-frame stop codons due to the 3' region of poly(A) tail, and constrained structure of original mRNA. These factors result in a poor yield of functional clones, which hinders the development of phage display cDNA libraries for decades. To solve these problems, Creative Biolabs has successfully established multiple unique techniques to generate high-quality cDNA libraries with large capacity, high density, and correct orientation.

Standard Procedure

The standard procedure to construct phage display cDNA libraries is performed in following steps:

• Extraction of RNA from indicated samples
• Synthesis of cDNA candidate pool
• Generation of phage-based cDNA libraries from size-selected cDNA
• Directional cloning of cDNA into any vector
• Production of a library with at least 10⁶ cfu
• Library amplification to ensure higher titer
• Verification of library quality by randomly picking clones and determining insert sizes by restriction digest
• Verification of library complexity by DNA sequencing of the inserts from a subset of randomly picked clones (Optional)

Diverse Types of cDNA Libraries

In addition, Creative Biolabs is confident in tailoring impeccable phage display cDNA libraries of diverse types, including:

Standard cDNA Library
In case urgent use for straightforward screening and downstream application is required, we can construct standard cDNA libraries with at least 3×10⁶ primary clones with an average insert size of at least 1 kb. We guarantee that all the clones are properly oriented for expression, antibody screening or isolation of specific cDNA clones.

Full-length cDNA Library
Our novel full-length cDNA synthesis procedure combines a proprietary process for reducing RNase H reverse transcriptase activity with unique techniques for mRNA isolation, 5' cap full-length enrichment, and reduction of oligo (dT) priming. Together, these steps greatly enhance the likelihood of constructing a library with full-length inserts, ensuring high success rate of subsequent screening.

Normalized cDNA Library
Special normalization procedures of Creative Biolabs can decrease the prevalence of clones with medium and high abundance, thus effectively eliminating biased sequencing. Therefore, normalized cDNA libraries can be a powerful tool for rare gene discovery.

Subtractive cDNA Library
This technique is exceptionally appropriate when the target gene is assumed to express in poor level. With proprietary self-subtraction and tissue-tissue subtraction techniques, we are able to reduce irrelevant abundant sequences 10-100 folds, making it easier to find desired targets.

In Creative Biolabs, either M13 or T7 phage system are available for cDNA library construction according to varied objectives. Meanwhile, we also provide cDNA library constructed via lambda phage vector as an alternative. Based on extensive experience and our versatile phage display platform, Creative Biolabs is specialized in constructing high-quality custom cDNA libraries. Scientists of Creative Biolabs will formulate a comprehensive and systematic plan using the most advanced techniques to deliver our clients' specific research goal.

Published Data

• Identification of Potential Protein Interactors for LEA Proteins Using Phage Display cDNA Libraries

Fig. 1 The three proteins recovered as independent clones among libraries, temperatures, and/or baits.¹

The study focuses on the identification of potential protein interactors for Late Embryogenesis Abundant (LEA) proteins using phage display cDNA libraries. The research reveals that LEA proteins, previously considered nonspecific molecular shields, may actually have specific protein targets they protect during stress conditions. The results demonstrate that phage display can successfully identify proteins that interact with LEA proteins, particularly those involved in protein synthesis and cellular stress responses. Phage display cDNA libraries in this study allows for the comprehensive screening of protein interactions, leading to the discovery of specific targets for LEA proteins. This enhances the understanding of the molecular mechanisms by which LEA proteins confer stress tolerance in plants, and could potentially lead to the development of crops with improved resilience to environmental stresses.

FAQ

1. What is a phage display cDNA library, and how is it constructed?

   A phage display cDNA library is a collection of cDNA sequences that are inserted into bacteriophages, allowing for the expression of these sequences as fusion proteins on the surface of the phage particles. The construction involves isolating mRNA from a specific tissue or organism, reverse transcribing it into cDNA, and then cloning these cDNA fragments into a phage vector. This library can then be used to identify protein-protein, protein-DNA, or protein-peptide interactions by exposing the library to target molecules and selecting phages that display binding interactions.

2. How does phage display cDNA library screening work?

   Screening a phage display cDNA library involves a process called biopanning, where the library is exposed to a target of interest, such as a protein or antigen. Phages that display peptides or proteins with high affinity for the target are bound and retained, while non-binding phages are washed away. The bound phages are then eluted, amplified, and subjected to further rounds of selection to enrich for phages that strongly interact with the target. Finally, the cDNA inserts from these phages are sequenced to identify the binding partners.

3. What are the applications of phage display cDNA libraries in biomedical research?

   Phage display cDNA libraries are applicated in the identification of novel protein-protein interactions, the discovery of receptor ligands, the mapping of epitopes, and the identification of proteins involved in disease processes. These libraries are also used to develop therapeutic antibodies, vaccines, and diagnostic tools by identifying binding partners with high specificity and affinity for disease-related targets.

4. What advantages do phage display cDNA libraries offer over traditional screening methods?

   Phage display cDNA libraries offer several advantages over traditional screening methods, such as yeast two-hybrid or co-immunoprecipitation. These advantages include the ability to screen vast libraries in a high-throughput manner, the potential to identify weak or transient interactions, and the flexibility to screen against a variety of targets, including proteins, peptides, and small molecules. Additionally, phage display allows for the direct selection of functional protein fragments and the identification of binding partners under a range of conditions.

5. How can phage display cDNA libraries contribute to drug discovery?

   Phage display cDNA libraries can significantly contribute to drug discovery by identifying protein targets or ligands that interact with drug candidates. By screening a cDNA library against a drug molecule, researchers can identify proteins that bind to the drug, providing insights into the drug's mechanism of action or potential off-target effects. Additionally, phage display can be used to develop peptide-based therapeutics by identifying peptides that bind to disease-related proteins with high specificity and affinity.

6. What challenges are associated with using phage display cDNA libraries?

   Using phage display cDNA libraries must ensure the quality and diversity of the library, which is crucial for successful screening. The construction of the library requires high-quality mRNA and efficient reverse transcription to generate full-length cDNAs. Another challenge is the potential for nonspecific binding during screening, which can lead to false positives. Careful optimization of biopanning conditions and the use of appropriate controls are necessary to overcome these challenges.

7. How is the diversity of a phage display cDNA library ensured?

   The diversity of a phage display cDNA library is ensured by using a broad and representative source of mRNA, such as from a specific tissue, developmental stage, or organism under particular conditions. The mRNA is reverse transcribed into cDNA, and the entire population of cDNAs is cloned into the phage vector. Maintaining this diversity during library construction is essential for increasing the likelihood of identifying meaningful interactions during screening.

8. Can phage display cDNA libraries be used to study protein interactions in specific tissues or developmental stages?

   Phage display cDNA libraries can be tailored to study protein interactions in specific tissues or developmental stages by isolating mRNA from the tissue or stage of interest. For example, a cDNA library can be constructed from mRNA isolated from developing seeds or stressed plants, allowing researchers to identify proteins that interact under specific physiological conditions. This approach is particularly useful for understanding tissue-specific or developmental stage-specific protein functions and interactions.

9. How do phage display cDNA libraries facilitate the identification of protein-protein interactions?

   Phage display cDNA libraries facilitate the identification of protein-protein interactions by enabling the display of a wide range of protein fragments on the surface of phages. These displayed proteins can interact with a target protein introduced during the screening process. Through rounds of selection, phages displaying proteins that bind to the target are enriched, allowing researchers to identify specific protein-protein interactions. This method is powerful for discovering novel interactors and understanding complex protein networks.

Resources

Use the resources in our library to help you understand your options and make critical decisions for your study.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.