Protease-stable Variants of Protein Domains Screening

For many applications, proteins must be stable for a long time and under a wide range of conditions, in particular, at the condition of protease. With more than a decade of experience in the field of phage display technology, Creative Biolabs is dedicated to meeting every unique need of our clients in Phage Display Library Construction and Phage Display Library Screening. Our seasoned scientists have developed a variety of strategies for the discovery of protease-stable variants of protein domains. We are pleased to introduce this high-quality service to all of our global customers.

The Background of Bacteriophage for Protease-stable Variants Identification

Bacteriophage M13 particles are stable without the loss of infectivity when incubated with proteases of proper concentration. The minor coat protein, protein III (pIII) of bacteriophage M13 has three domains: C-terminal (CT) domain and two N-terminal domains (N1 and N2). Phage particles cannot infect bacterial cells if their N-terminal domains are absent or removed. This advantage could be used to isolate variants with resistance to speciﬁc proteases. When the phage display protein variants library is subjected to the pressure of protease treatment, protein variants that remain active will be enriched during each subsequent round of selection.

Fig.1 Schematic drawing of a phage hosting a protein (P) between CT and N2 of the gene-3-protein and the selection procedure (Sieber et al. 1998).

Strategies for the Identification of Protease-stable Variants in Creative Biolabs

Based on our powerful phage display platform, scientists of Creative Biolabs have developed a variety of strategies for the discovery of protease-stable variants. One strategy is to clone the protein variants between the N2 and CT domains of the pIII and subjected to protease treatment. Protein variants susceptible to protease will be cleaved, resulting in loss of the N-terminal domains, and thus making the phage noninfectious. Such clones will be eliminated from the library. If the protein variants are resistant to protease, the protein III remains intact during the selection process, and these phage particles will be isolated and enriched during each subsequent round of selection. Another strategy is that protein variants are subcloned in-frame with the gene III coding sequence and an N-terminal afﬁnity tag. Variants that are cleaved by the protease lose their afﬁnity tag and then removed during the screening. Protease-resistant variants retain their N-terminal afﬁnity tag and are afﬁnity captured and enriched.

Key Advantages of Protease-stable Variants Discovery Service

A variety of proteases can be used, including trypsin, chymotrypsin, pepsin, endoproteinase Glu-C, proteinase K, etc.
Diverse strategies for library construction
Tailored biopanning protocol
High sensitivity and high-throughput
Advanced phage display platform

Powered by our advanced phage display platform and professional scientific staff, Creative Biolabs is fully competent and dedicated to providing one-stop-service for the discovery of protease-stable variants, from phage display protein variant library construction, library screening to the validation of specific variants. For more detailed information, please feel free to contact us or directly send us an inquiry.

Reference

Sieber, V.; et al. Selecting proteins with improved stability by a phage-based method. Nat Biotechnol. 1998, 16(10):955-60.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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