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Alanine scanning is a precise and systematic method in protein engineering for identifying key amino acid residues by replacing them with small, chemically stable alanine. This approach reveals each residue's contribution to structure, stability, activity, and ligand binding, enabling the mapping of active sites and functional regions. Compared with random mutagenesis, alanine scanning offers high controllability and comprehensive coverage, preserving overall protein conformation, supporting functional analysis, antibody optimization, drug target discovery, and rational protein design, and providing high-resolution functional maps for complex proteins.
Creative Biogene leverages a state-of-the-art molecular biology and protein engineering platform combined with extensive library construction experience and advanced synthesis strategies to deliver high-quality, customizable alanine scanning libraries. Our services cover the entire workflow from experimental design and library construction to quality control and results submission, ensuring comprehensive coverage, high accuracy, and reproducibility that meet international standards.
The key to library construction lies in rational design and coverage of each mutation site. Creative Biogene develops optimized alanine scanning schemes based on the target protein sequence, structural information, and functional requirements. By integrating sequence conservation, domain features, active sites, and potential interaction interfaces, we select residue substitutions that maximize functional residue identification while minimizing effects on protein conformation. Library diversity and balance are also considered, with predictive algorithms assessing structural and functional impacts to ensure full coverage.
During construction, advanced oligonucleotide synthesis, PCR amplification, and targeted vector insertion enable precise single-residue alanine substitutions. PCR and enzymatic conditions are optimized to maximize on-target mutation efficiency while minimizing off-target errors. Linear DNA libraries are validated and cloned into specified vectors to create high-quality plasmid libraries for protein expression, functional screening, and activity assessment. Library diversity and coverage are rigorously quantified, ensuring a representative sampling of each residue's effect.
1Linear DNA Library Synthesis and Validation: High-fidelity PCR amplification is used to introduce alanine substitutions at each target residue. Randomly selected 12–96 clones are sequenced to confirm library diversity and accuracy.
2Plasmid Library Construction: Linear DNA libraries are cloned into specific vectors, and random clone sequencing is performed to ensure library completeness and precision.
3Library QC and Data Submission: Clients receive 1–2 μg of linear DNA library or 5–10 μg of plasmid library in lyophilized form, accompanied by raw sequencing chromatograms, electrophoresis images, restriction digestion validation, sequence alignment data, and COA files, ensuring immediate applicability for downstream experiments.
Creative Biogene provides full technical support throughout the project, addressing experimental design, library construction, and quality control issues. Each project is assigned a dedicated project manager to provide regular updates, report experimental progress, and ensure clients maintain full visibility over each step. Whether clients are performing alanine scanning for the first time or conducting high-throughput screens, we provide expert guidance and tailored solutions.
Alanine scanning libraries have broad applications, enabling deep insights into protein function and molecular mechanisms:
These applications are valuable for both basic research and biopharmaceutical development, including antibody optimization, enzyme engineering, vaccine design, and therapeutic protein development.
Creative Biogene combines mature platform technologies, standardized workflows, and extensive library construction experience. Clients benefit from high-quality, customizable alanine scanning libraries with comprehensive QC data and expert support, including:
Through Creative Biogene's alanine scanning library service, researchers can systematically map critical residues, optimize protein engineering strategies, and accelerate drug discovery and molecular mechanism studies. For more information or to request a customized alanine scanning library, please contact our technical team via Consultation.
1. Can the library cover the entire protein sequence?
Yes. Creative Biogene's alanine scanning library strategy enables systematic replacement of every non-alanine residue in the target protein, ensuring comprehensive coverage. Feasibility and library balance are evaluated during the design phase.
2. How is library accuracy ensured?
We employ high-fidelity PCR, stringent enzymatic treatment, and sequencing validation. Both linear DNA and plasmid libraries are randomly sampled and sequenced to confirm mutation accuracy and preserve non-target regions. Overall accuracy can reach 100%.
3. Can the library be used directly for expression and screening?
Yes. Constructed plasmid libraries are ready for protein expression, functional assays, and enzymatic analysis. QC reports and COA files ensure smooth downstream applications.
4. Why is alanine scanning performed using substitutions to alanine rather than other amino acids?
Alanine scanning is commonly used in protein structure studies because it replaces the side chain's reactive group with a small, non-functional methyl group, causing minimal impact on protein structure. In contrast, glycine has an even smaller side chain, but its alpha carbon lacks chirality, which can significantly affect protein structure, so it is generally not used.
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