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Identification of Functional Protein Regions Through Chimeric Protein Construction
Published on: January 8, 2019
Random dissection to select for protein split sites and its application in protein fragment complementation.
Yong Chen1, Shuang Li, Tingjian Chen
1Department of Chemical Engineering, Tsinghua University, Beijing, China.
Protein Science : a Publication of the Protein Society
|January 24, 2009
Summary
This study introduces a novel chloramphenicol acetyl transferase (CAT) selection method for efficiently identifying protein split sites. This technique aids in designing protein fragments for improved protein complementation assays and directed evolution.
Area of Science:
- Molecular Biology
- Biochemistry
- Protein Engineering
Background:
- Identifying protein split sites is crucial for protein fragment complementation assays (PCA).
- Existing methods can be time-consuming and inefficient for discovering optimal fragment pairs.
Purpose of the Study:
- To develop and evaluate a rapid selection procedure for identifying protein split sites using chloramphenicol acetyl transferase (CAT) as a reporter.
- To apply this method to guide the design of protein fragments for PCA and improve protein activity through directed evolution.
Main Methods:
- A selection procedure utilizing CAT reporter to screen folded protein fragments from random gene digestion and reassembly libraries.
- Application of the CAT-based protein random dissection protocol to aminoglycoside-3'-phosphotransferase I (APH(3')-I) for PCA.
- Optimization of identified protein fragments using error-prone PCR and directed evolution.
Main Results:
- The CAT selection procedure efficiently identified protein fragments with controllable lengths, validated by tryptophan synthase alpha subunit (TSalpha) and TEM-1 beta-lactamase.
- Three nearly bisectional sites and multiple split points were identified in APH(3')-I, leading to the discovery of novel fragment pairs.
- Three of four tested APH(3')-I fragment pairs partially restored enzyme activity, and a truncated active form (Delta1-25) was found.
- Directed evolution of a weakly active APH(3')-I fragment resulted in a fourfold activity improvement after one round of PCR.
Conclusions:
- Protein random dissection based on CAT selection is an efficient strategy for finding protein breakage points.
- This method effectively guides the design of protein fragments for PCA and facilitates the discovery of more active fragment pairs.
- Classical directed evolution can further enhance the activity of engineered protein fragments identified through this approach.
