Constructing a protein mutation library is a key step to increase protein production, improve function, or study structure-function relationships, which can generate mutant proteins with improved or new properties. As a leading protein production and engineering expert, CD Formulation provides a wide range of protein mutation library construction services. Our team of experts has extensive expertise in constructing custom mutagenesis libraries and can provide a variety of strategies to create your diverse protein mutation library, including gene synthesis, error-prone PCR, degenerate codon utilization, and DNA shuffling.

What is Protein Mutation Library Construction?

Protein mutation library construction refers to the process of creating a collection or library of specific protein variants, which is usually used to study how changes in protein sequence affect its structure, function, or interaction with other molecules. This technology has been widely used to optimize protein function by identifying and modifying key residues to change the structure and function of proteins. It is an important method of protein engineering for generating new proteins with higher solubility, stability, expression level, etc. The construction of mutant libraries provides a method to obtain large and diverse proteins with desired complexity and has become a powerful, high-throughput tool for optimizing protein structure and function.

Fig. 1 Overview of the mutant library screening strategy. (Tavares D, et al., 2019)

Explore Our Therapeutic Protein Mutation Library Construction Service

Protein mutant libraries have important applications in determining the important amino acid residues that control physiological activities and pathological processes. By screening protein mutant libraries, new components of signaling pathways or receptors can be identified and excellent target proteins can be created.

CD Formulation's strong expertise in high-throughput gene synthesis and site-directed mutagenesis enables us to construct complex protein mutant libraries to accelerate your projects. We offer multiple strategies to build your diverse protein icon library, including gene synthesis, mutagenesis, DNA shuffling, pilot-scale protein expression, and final protein purification.

Site-Directed Mutagenesis Library Construction

Site-directed mutagenesis library construction is used to introduce mutations at specific positions to study gene function, protein structure-function relationship, etc. Our scientists combine de novo gene synthesis and site-directed mutagenesis technology to provide the most advanced site-directed mutagenesis library construction services. Our site-directed mutagenesis libraries are constructed by replacing any given residue with any other 19 common amino acids.

Scanning Point Mutation Library Construction

Scanning point mutation is a method to systematically improve protein performance, allowing for improving the standard alanine/cysteine scanning by replacing each amino acid with all 20 amino acids at the same time. For each codon of interest, a small site-saturated library is constructed. The library can be provided as a pool or in individual formats of any substitution variant (19 in total).

Random or Degenerate Library Construction

With advanced degenerate oligonucleotide and in vitro library synthesis technology, we can introduce random mutations in synthetic DNA fragments with maximum flexibility and generate any form of full-length gene randomization or degeneration, thus achieving highly precise randomization within oligonucleotides.

Workflow for Therapeutic Protein Mutation Library Construction

Fig. 2 Workflow for therapeutic protein mutation library construction. (CD Formulation)

• Library Design: The process involves creating mutations in the gene sequence of the target protein. Mutations can be point mutations (single amino acid changes), insertions, deletions, or more complex alterations. Designs can be random (to explore a wide range of changes) or targeted (focusing on specific regions of the protein known to be important for function).
• Variant Synthesis: Mutant genes encoding protein variants are synthesized. This can be done using techniques such as site-directed mutagenesis, polymerase chain reaction (PCR), or through synthetic biology approaches.
• Transformation and Expression: The mutant gene sequence is then introduced into a suitable host cell (e.g., bacteria, yeast, or mammalian cells) for expression. The host cell is transformed with a plasmid carrying the mutant gene, resulting in the production of the corresponding protein variant.
• Screening and Selection: The expressed protein is then screened for desired properties, such as improved stability, activity, binding affinity, or resistance to inhibitors. High-throughput screening methods can be used to rapidly evaluate large numbers of variants.
• Variant Characterization: Promising variants that exhibit the desired properties are further characterized and analyzed to understand their structure and function.
  • Structural Studies: Techniques such as X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy are used to elucidate the 3D structure of protein variants to gain an intuitive understanding of how mutations affect folding and stability.
  • Biochemical Assays: Enzyme activity assays or binding studies are used to determine how mutations affect the biological function of the protein.
  • Stability Tests: Thermal stability assays or stress tests, e.g., exposure to denaturants or proteolytic enzymes, reveal how mutations affect the stability of the protein under various conditions.
• Optimization and Iteration: Based on the characterization results, the best-performing variants are screened for further optimization. This may involve additional rounds of mutagenesis and screening to improve the desired properties, creating a more targeted library of variants.

Why Choose Our Therapeutic Protein Mutation Library Construction Service?

• Our team consists of highly skilled scientists with extensive experience in protein engineering and molecular biology, excelling in utilizing industry best practices and cutting-edge technologies to develop optimized mutation libraries tailored to your specific needs.
• We employ advanced methods to generate libraries with high mutation diversity, ensuring a wide range of variants. This diversity increases the chances of identifying therapeutic candidates with desirable properties.
• Our Protein Mutation Library Construction service has a high success rate, allowing for the receipt of all possible truncation variants with a library diversity of up to ~40,000.
• We utilize the latest high-throughput sequencing and gene synthesis technologies to ensure high accuracy and reliability of library construction.
• We employ rigorous validation methods to ensure the integrity and functionality of the mutation library.
• Our services are customizable, allowing you to specify the parameters and goals of library construction to better meet your research goals.

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CD Formulation's therapeutic protein mutation library construction service can efficiently provide you with thousands of protein variants. Please don't hesitate to contact us if you are interested in our services. We look forward to cooperating with you.