Incorporation of Unnatural Amino Acids into Proteins with mammalian epxression systems

Background

In the field of biotechnology and pharmaceuticals, protein engineering has become a key technical means for the innovation and development of new therapeutic drugs. Among them, the introduction of unnatural amino acids (UAAs) to modify or enhance protein function has become the forefront of current research. Traditional protein synthesis is limited to 20 standard amino acids, while the introduction of unnatural amino acids gives proteins new functional properties, such as fluorescent labeling, photocrosslinking and bioorthogonal reaction groups, which greatly expands the application range of proteins, including but not limited to drug development (such as site-specific coupling ADC drugs), protein-protein interaction research, structural biology and biosensor design.

Mammalian cell expression systems are considered to be ideal platforms for expressing proteins containing unnatural amino acids because they can achieve post-translational modifications similar to human proteins (such as glycosylation, phosphorylation, etc.). Compared with E. coli or yeast expression systems, mammalian cells (such as HEK293 and CHO cells) can better ensure the correct folding and functional integrity of complex proteins, especially for the study of antibodies, membrane proteins and secreted proteins. Using advanced genetic code expansion technology, unnatural amino acids are efficiently inserted into specific sites of target proteins through the orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) system. A variety of UAAs are available, including but not limited to p-acetylphenylalanine (pAzF), azidolysine (AzK) and photo-crosslinked amino acids (such as DiZPK) to meet diverse research needs, providing a flexible and powerful toolkit to support cutting-edge scientific research and technology development. This not only improves the depth and breadth of research, but also opens up new avenues for the development of new therapeutic strategies.

Service Procedure

Profacgen is committed to providing customers with a comprehensive one-stop service experience, covering the entire process from the design and synthesis of unnatural amino acids, the construction of mammalian cell lines, the expression and purification of target proteins, to the final product quality testing. At the same time, we also provide detailed data analysis and technical support to ensure that customers' projects can be carried out efficiently and smoothly.

Services we provide:

Vector construction: Custom mammalian expression vectors containing UAA insertion sites.

Cell line development: Establish HEK293 or CHO cell lines that stably express orthogonal tRNA/aaRS.

Unnatural amino acid selection: Provide testing and optimization of multiple UAA or customer-specified UAA.

Protein expression and purification: Large-scale expression of UAA-containing proteins, and affinity purification, SEC or HPLC analysis.

Functional validation: Confirm the correct incorporation of UAA and protein function through mass spectrometry, Western blot, activity detection, etc.

Figure 1. Service Process.

Our Advantages

• Highly specific integration: Adopting an optimized orthogonal tRNA/aaRS system to ensure the precise insertion of unnatural amino acids (UAA) at the target site.
• Flexible unnatural amino acid selection: Providing a variety of UAAs and supporting customers to customize rare amino acids to meet the needs of photocrosslinking, click chemistry and other applications.
• Strict quality control: Verifying the UAA incorporation site and efficiency through LC-MS/MS, and providing purity analysis reports such as SDS-PAGE and SEC-HPLC.
• Mature one-stop service: Covering the entire process from vector design, cell line construction to protein purification, shortening the project cycle and reducing the trial and error costs of customers.

FAQs

Please contact us to provide more detailed information for evaluation.