Construction of Polypeptide Library

The yeast two-hybrid random peptide library technology is a two-hybrid screening system that uses a random peptide library as a screening target. Lifeasible constructs yeast expression vectors by fusing random DNA fragments with GAL4AD, and a screenable and amplifiable peptide library is formed. It can be applied to developing novel peptide drugs and diagnostic reagents and studying protein structure and function.

Random 16 Peptide Library Yeast Two-hybrid Vector Construction

We have rich experience in library construction; we can provide you with a random 16-peptide library yeast two-hybrid vector construction service. We amplify the synthetic random DNA template by PCR, clone it into yeast expression plasmid pGADTGH after double digestion, construct yeast two-hybrid random cyclic peptide library and check its library capacity and randomness, amplify, extract and purify yeast two-hybrid cyclic peptide library plasmid. Finally, the yeast two-hybrid random cyclic peptide library was successfully constructed, and a large number of high-purity cyclic peptide library plasmids were amplified.

Library volume is the main indicator of random peptide libraries. Currently, we use the most efficient electro-conversion method for library construction, up to 10⁹-10¹⁰/μg. For most protein interaction interfaces, only 3-5 key residues are sufficient to ensure sufficient interaction stability. There is a high probability that this series of clones may appear in the library. The longer the peptide, the better it is to find epitope sequences with affinity, especially for binding epitopes of target molecules where the key amino acids are not contiguous.

Construction of a Two-promoter Yeast Random Peptide Library for Screening Proteolytic Peptides

We took advantage of the spatially separable DNA binding domain (BD) and transcriptional activation domain (AD) of yeast transcriptional activator, which can be inserted downstream of the BD domain of pBridge with X gene, downstream of the second conditional methionine promoter (PMET25) with a random 10-peptide library with thioredoxin (TrxA) as the backbone, and inserted the Y gene into the AD domain containing pACT2, co-transforming yeast host cells. Thus, the expression of the nutrient-deficient phenotype and the corresponding reporter gene can be used to screen for small peptide molecules that promote X-Y dissociation.

• We chose the dissociation of protein interactions as the entry point and combined the conformational random peptide library with the triple hybridization system to achieve a truly high-throughput large-scale screening.
• Based on obtaining the genes of two known interacting proteins, we can directly screen the dissociated peptides without purifying the proteins, eliminating the tedious process of immunoscreening and realizing the complementary advantages of peptide chemistry and molecular biology.
• The screening target is changed from a single protein to an interacting protein complex, and the dissociation reaction in eukaryotic cells is closer to the natural state than the screening done in vitro.
• Peptides displayed through conformationally random peptide libraries are more conducive to screening for high-affinity ligands due to their restricted conformation.

Our service offers new ways for our clients to study the dissociation of protein complexes. The obtained dissociated peptides can effectively block essential protein complexes in signaling pathways, which can provide drug targets for treating diseases caused by abnormal signal transduction pathways, such as autoimmunity and tumors.

Technical Advantages

• No purification of target proteins is required.
• Closer to the physiological state of mammalian cells than prokaryotic cells, better able to maintain the natural conformation and function of target proteins.
• The screening of yeast two-hybrid system occurs in yeast cells, which is more likely to maintain the natural conformation and function of the target protein for intracellular proteins. The peptides obtained from such screening are more likely to be biologically active.
• It has high sensitivity and can detect weak and transient interactions.

For more information, please feel free to contact our staff.