Creative BioMart is a well-known expert who is committed to incorporating a variety of non-canonical amino acids into proteins to introduce new functions, redesign natural proteins and artificial protein macromolecules. With years of experience, we provide customized services for engineering fluorescent proteins with new properties to precisely meet customer requirements.

Introduction of Fluorescent proteins

Fluorescent proteins are members of a structurally homologous class of proteins that have the unique property of being self-sufficient, forming visible-wavelength chromophores from 3 amino acid sequences within their own polypeptide sequence. With the development of biotechnology, scientists tagged fluorescent protein (GFP) and its variants on many proteins to visualize biological processes. Engineered fluorescent proteins have been widely used in biological research to study gene expression, protein function and trafficking, and protein-protein interactions. In addition, fluorescent proteins have also been designed as biosensors to detect intracellular signaling molecules and other small-molecule metabolites.

Fig 1. The two basic designs of Fluorescent protein-based biosensors containing non-canonical amino acids. (Roman-Arocho G M, et al., 2020)

Services

Fluorescent proteins are traditionally modified with 20 typical amino acids, which limits the number of functional groups that can be used to design and construct novel fluorescent proteins. The large size of native fluorescent proteins may interfere with the structure and function of tagged proteins. Current efforts are focused on developing small fluorophores that enable detection of the structure and/or location of target proteins in vivo with minimal interference. As a leading service provider of protein engineering, Creative BioMart has successfully expanded the genetic code by incorporating non-canonical amino acids, modifying the chemical and physical properties of fluorescent proteins.

We provide a one-stop service for fluorescent proteins for the specific incorporation of non-canonical amino acids into fluorescent proteins in living cells, including:

• Fluorescent protein mutagenesis service: we replace the chromophore Tyr66 with a tyrosine analog to generate new fluorescent proteins. And the Tyr66 codon was mutated to Amber nonsense codon by overlapping PCR or the commercial QuikChange kit.
• Co-transformation service for E. coli host cells: we use electroactive cells or chemically active cells as hosts. Amber nonsense codon-mediated translation termination was abolished in E. coli strains.
• Fluorescent protein expression service: we use the pBK/pLei system to induce fluorescent protein expression by adding ideal non-canonical amino acids.
• Fluorescent protein characterization service: we use the Bradford method and SDS-PAGE to determine the concentration and purity of fluorescent proteins containing unconventional amino acids. Determine their emission and excitation wavelengths by fluorometer or fluorescence microplate reade.

In addition, we can design fluorescent proteins incorporating non-canonical amino acids as sensors for selective detection and quantification of small molecules (such as H₂O₂, H₂S, Ca²⁺, Cu²⁺, Fe²⁺, and Hg²⁺).

Creative BioMart is committed to engineering fluorescent proteins to provide a variety of new properties for use as tools for tracking gene expression, visualizing target proteins, and studying protein-protein interactions. We will work with you to develop the most appropriate strategy and provide the most meaningful data for your research for accelerating the research of life sciences. If you are interested in our services, please do not hesitate to contact us for more information.

Reference

1. Roman-Arocho G M, Shang X, Niu W, et al.. (2020) Design of fluorescent protein-based sensors through a general protection-deprotection strategy[J]. Methods in enzymology. 640: 63-82.

• Protein Engineering Based on Backbone Amides
• Protein Engineering Based on Fluorinated Amino Acids
• Protein Engineering Based on Photoreactive Amino Acids