Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain.


Protein libraries based on natural scaffolds enable the generation of novel molecular tools and potential therapeutics by directed evolution. Here, we report the design and construction of a high complexity library (30 x 10(13) sequences) based on the 10th fibronectin type III domain of human fibronectin (10FnIII). We examined the bacterial expression characteristics and stability of this library using a green fluorescent protein (GFP)-reporter screen, SDS-PAGE analysis, and chemical denaturation, respectively. The high throughput GFP reporter screen demonstrates that a large fraction of our library expresses significant levels of soluble protein in bacteria. However, SDS-PAGE analysis of expression cultures indicates the ratio of soluble to insoluble protein expressed varies greatly for randomly chosen library members. We also tested the stabilities of several representative variants by guanidinium chloride denaturation. All variants tested displayed cooperative unfolding transitions similar to wild-type, and two exhibited free energies of unfolding equal to wild-type 10FnIII. This work demonstrates the utility of GFP-based screening as a tool for analysis of high-complexity protein libraries. Our results indicate that a vast amount of protein sequence space surrounding the 10FnIII scaffold is accessible for the generation of novel functions by directed as well as natural evolution. Study holds ProTherm entries: 22714, 22715, 22716, 22717, 22718, 22719 Extra Details: BC and FC loop sequnces are, VGVPPTL and FDRLKAIYTE, respectively. 10FnIII; combinatorial protein library; in vitro selection; GFP reporter screen

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:53 p.m.

Version: 1

Publication Details
Olson CA;Roberts RW,Protein Sci. (2007) Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain. PMID:17322532
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Fibronectin P02751 FINC_HUMAN
96.8 Fibronectin P07589 FINC_BOVIN
94.0 Fibronectin Q28275 FINC_CANLF
92.1 Fibronectin Q28377 FINC_HORSE
90.9 Fibronectin Q91400 FINC_NOTVI