The demand for stable proteins has surged with the growing applications in biological research and clinical treatments. Stable proteins possess various crucial characteristics, including the ability to maintain stability even in highly concentrated solutions. This unique feature enables the study of protein-protein interactions kinetics and facilitates three-dimensional structure determination using techniques like NMR or X-ray crystallography. Moreover, stable proteins serve as exceptional scaffolds for generating affinity reagents, essential for targeting specific proteins of interest. At Creative Biolabs, we leverage our extensive field experience and phage display platform to provide comprehensive services for the development of stable protein domain variants.

Stable Variants of Protein Domains

Screening for stable variants of protein domains through phage display technology is apivotal method to obtain these stable protein variants. Phage M13's Protein III, also known as the secondary shell protein, consists of three structural domains: one C-terminal structural domain and two N-terminal structural domains (N1 and N2). Removal of the N-terminal structural domain renders the phage particle unable to infect bacterial cells. This property is harnessed to isolate variants with enhanced properties such as improved thermodynamic stability and resistance to specific protease classes. Library variants are subcloned between the N2 and CT domains of Protein III and subjected to thermal and protease treatment. Protease-sensitive variants will be cleaved, resulting in the loss of the N-terminal structural domain and thus rendering the phage non-infectious. Consequently, these clones are eliminated from the library. Protease-resistant variants, on the other hand, retain their structural integrity during the selection process. These phage particles are isolated, propagated, and enriched in subsequent rounds of selection. Additionally, variants subcloned in-frame with gene III coding sequences and N-terminal affinity tags lose these tags if cleaved by protease. Phages lacking affinity tags are not captured by affinity chaperones (such as Ni-NTA for tagged proteins, streptavidin for biotinylated proteins, and epitope-tagged antibodies) and are removed during the selection process. Proteins, proteases, or denaturants denatured or cleaved by heat do not bind to conformation-specific antibodies, interacting proteins, or ligands and are consequently eliminated from the library. Variants that remain folded and active are retained and enriched. Our screening process for protease-stable variants of protein domain can be customized according to specific requirements, ensuring tailored results for every client.

Heat stabilization offers numerous practical advantages for proteins. Firstly, proteins can maintain their structure and activity even at high temperatures. Secondly, at room temperature, a higher proportion of proteins retain their folded state. Thirdly, heat-stabilized proteins tend to accumulate in larger quantities within E. coli cells. Additionally, these proteins are often more soluble at high concentrations, as they adopt stabilized structures that resist aggregation. Consequently, screening for thermal stable variants of protein domains becomes crucial in various applications. In the screening process, Phage M13 solutions were exposed to a temperature of 65°C for 15 minutes without losing infectivity. However, the preservation of protein structural domains depends on their inherent thermal stability rather than Protein III or virus particles. For instance, variants of the Bacillus subtilis endoxylanase (XynA) protein displayed on phages were subjected to elevated temperatures during affinity selection steps. After three rounds of screening, mutations enhancing thermal stability were identified. These mutations raised the half-inactivation temperature by 2-3 °C compared to the wild-type enzyme. Notably, these mutations were not previously identified in conventional engineering studies. Some examples of resistance to post-heating aggregation include single-domain human antibody variable domains binding to protein A or chicken egg lysozyme. It's important to highlight that besides random mutagenesis and phage selection, computational methods can also be employed to identify protein variants with improved stability when the three-dimensional structure is known, computational methods can also be used. This approach involves replacing specific amino acid residues predicted to cause thermolability due to their significant shifts with smaller residues, facilitating better packing.

More Details on Stable Variants of Protein Domains Discovery by Phage Display

Introduction to Thermal Stable Variants of Protein Domains Screening by Phage Display
Introduction to Protease-stable Variants of Protein Domains Screening by Phage Display

At Creative Biolabs, we possess extensive knowledge and experience in stable variants of protein domains. Our expertise allows us to offer tailored solutions for various applications.

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