In vitro molecular optimization is a very efficient means of generating mutant proteins with improved or novel properties, identifying regulatory sequences, and probing for structurally and functionally critical residues. Mutant libraries constructed using the in vitro molecular optimization method provide a useful approach to the systematic study of protein properties, regulation, and function.
Creative Biolabs' strong expertise in de novo gene synthesis allows us to synthesize complex protein libraries without any dramatic increase in cost. Our mutant library services include site-directed mutagenesis libraries, sequential permutation scanning libraries, and randomized and degenerated libraries.
Site-Directed Mutagenesis Libraries
Creative Biolabs combines its expertise in de novo gene synthesis and site-directed mutagenesis into an excellent site-directed mutagenesis library construction service. The site-directed mutagenesis library offers a great platform for protein function and active center studies. In these libraries, any given residue can be substituted with any of other 19 common amino acids, creating systematic combinations of amino acid mutations that reveal any significant pattern.
Scanning Point Mutation Libraries
Scanning point mutation is a systematic means of improving protein performance. It outperforms standard alanine/cysteine scanning by replacing each amino acid with all 20 amino acids simultaneously. This technique provides a detailed profile of each amino acid at the position. For each codon of interest, a small, site-saturated library is constructed. This library can be delivered as a pool or in a separated format for any substitution variant (19 in total). The application of Creative Biolabs' expertise in de novo gene synthesis to the field of sequential permutation scanning allows us to provide superb sequential permutation scanning library construction services.
The key point in protein array technology is that arrayed proteins should be correctly folded, functional and of the greatest flexibility for experimental design. For this purpose, the proteins spotted on our arrays were all expressed by yeast cells in a secreted form and purified under native conditions. These protein arrays support a broad range of applications and enable rapid profiling of thousands of biochemical interactions. Highly sensitive and reproducible results are routinely obtained by our staff scientists during custom services. In addition, comprehensive Bioinformatics support including GO analysis and Pathway analysis is included in our custom protein array services.
Randomized and Degenerated Libraries
With our advanced degenerated oligonucleotide techniques, Creative Biolabs can generate any form of randomization or degeneration of the full-length gene in a synthetic DNA fragment. This permits controlled, highly precise randomization within oligonucleotides.
Creative Biolabs' in vitro library synthesis technology can introduce random substitutions on a controlled level with maximum flexibility. The mutation frequency can be set at any value between 1 and 20 mutations per kb. A peer group of 48, 96, or 192 individual transformants is sequence-verified.
Truncation technology incrementally trims genes around a defined core region. Genes can be truncated from both the 5' and the 3' ends simultaneously to produce an incremental truncation library. Unlike other methods, our process preserves the open reading frame, avoiding out-of- frame mutations.
• Optimize solubility
• Evaluate minimal functional-protein size
• Identify functional domains
• Screen for inhibitors
• Map epitopes
• No system-based out-of-frame mutations
• Low ancillary mutation rates—>90% sequence integrity (depending on length of the gene;
• unintended mutants will have amino acid substitutions in most cases)
• High success rates—receive all possible truncation variants; library diversity of up to ~40,000