Efficient Cloning of Inserts for Phage Display by Golden Gate Assembly

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Engineering

Background:

• Phage display is a powerful technique for identifying high-affinity binders.
• Traditional cloning methods for phage display can be cumbersome.
• Efficient genetic manipulation is key to optimizing phage display libraries.

Purpose of the Study:

• To develop an improved cloning strategy for phage display.
• To enable efficient insertion of genetic sequences into the N-terminus of phage protein III.
• To demonstrate the versatility of the new method for displaying various protein tags and binders.

Main Methods:

• Utilized a novel vector with kanamycin resistance and BsaI sites.
• Employed Golden Gate cloning for seamless DNA insertion.
• Targeted the N-terminus of mature protein III for genetic integration.
• Performed pentavalent display of multiple inserts.

Main Results:

• Successfully implemented Golden Gate cloning into the N-terminus of protein III.
• Demonstrated pentavalent display of six distinct inserts.
• Confirmed the successful display of AviD-tag, Z-domain of protein A, Myc-tag, ALFA nanobody, BC2 nanobody, and Flag-tag.

Conclusions:

• The new Golden Gate cloning vector and method streamline the construction of phage display libraries.
• This approach allows for efficient and versatile display of diverse functional proteins on phage particles.
• The method facilitates the discovery of novel high-affinity binders for various applications.