Construction of yeast hybrid library: Gateway technology-Yeast Display Technology Special Topic-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Construction of yeast hybrid library: Gateway technology

Background

A yeast library functions as a repository for a large number of exogenous fragments. Using yeast cells as vectors, genes expressing exogenous proteins are inserted to form a large collection. Such libraries are indispensable tools in biological research, enabling studies on gene function, interactions, and other aspects.

Through yeast libraries, one can investigate the regulatory networks governing life processes, discover new protein pathways, and advance experimental progress. Yeast hybridization technology is widely recognized as a classic approach for studying molecule-protein and protein-protein interactions.

As a fungus, yeast offers unique advantages: its eukaryotic characteristics and suitability for industrial-scale production make it widely applicable in life science research. Two main methods are used for constructing yeast libraries: the Gateway method and the SMART method. These techniques address different needs and conditions. This article focuses on the Gateway method.

Principle

The Gateway method is based on the properties of the site-specific recombinase encoded by bacteriophage λ (int), which can integrate and excise the viral genome from the host chromosome. Int executes a highly directed, tightly regulated reaction that depends on the auxiliary DNA-bending protein IHF (Integration Host Factor)^[1]. This precise regulation enables both the integration and excision recombination pathways, which are opposite reactions that are essentially irreversible under distinct physiological and environmental signals. As illustrated: with the assistance of IHF, the λ integrase (Int) performs recombination between the supercoiled attP site and the linear attB site, generating an integrated phage chromosome containing attR and attL sites. Through the action of Int, Xis (phage-encoded excisionase), IHF, and Fis protein, the integrated DNA is excised, regenerating attP and attB.

Figure 1 Working model of phage integration and excision with the host genome

Based on this mechanism, DNA fragments (e.g., genes) located between recombination sites can be mixed in vitro with another new vector containing compatible recombination sites and incubated with the λ integrase recombination proteins, transferring the gene into the new vector. The Gateway method comprises two major components: the construction of an entry clone, and the subsequent recombination of the entry clone into an expression vector (Figure 2B).

Entry clone (Figure 2C): Recombination sites attB1 and attB2 are added to the gene fragment by PCR. This fragment is incubated with a donor vector containing attP1 and attP2 sites in the presence of λ integrase (Int) and the auxiliary integration host factor IHF. Upon recombination and integration, the inserted gene in the vector becomes flanked by new recombination sites, attL1 and attL2.

Recombination into the expression vector (Figure 2D): The plasmid containing the integrated gene flanked by attL1 and attL2 sites is co-incubated with an expression vector (yeast expression vector) containing attR1 and attR2 sites. Purified λ Int, Xis, and IHF proteins are added to the reaction to facilitate recombination of the gene fragment into the expression vector. Recombination reactions can only occur between B1 and P1, B2 and P2, L1 and R1, and L2 and R2 to ensure correct DNA orientation.

Both vectors contain the ccdB gene between the att sites (P1-P2 and R1-R2). The expression product of the ccdB gene disrupts DNA gyrase. When the plasmid is successfully constructed and the genome is properly integrated, ccdB is replaced and the vector can survive. Finally, the resulting plasmid is electroporated into yeast cells for preservation.

Figure 2 Schematic diagram of the Gateway principle ^[2]

TekBiotech (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform based on phage display and yeast display technologies. We provide high-quality monoclonal antibody development services for scientists worldwide, covering various formats including scFv, VHH, and Fab. Furthermore, we can develop antibodies with diverse functional and structural characteristics, including but not limited to neutralizing antibodies, conformation-specific antibodies, and cross-reactive antibodies. Additionally, TekBiotech offers supporting downstream services such as antibody expression validation, antibody humanization design and validation, antibody affinity maturation, and CAR-T candidate sequence design, meeting the diverse needs of clients for antibody drug development.

References

[1] Landy A. The λ Integrase Site-specific Recombination Pathway. Microbiol Spectr. 2015;3(2):MDNA3-0051-2014.

[2] Walhout AJ, Temple GF, Brasch MA, et al. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 2000;328:575-92.

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