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


Abstract

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

ID: cYDF2M6E3

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 PDB Entries

Structure ID Release Date Resolution Structure Title
1E88 2000-09-18T00:00:00+0000 0 Solution structure of 6F11F22F2, a compact three-module fragment of the gelatin-binding domain of human fibronectin
1E8B 2000-09-18T00:00:00+0000 0 Solution structure of 6F11F22F2, a compact three-module fragment of the gelatin-binding domain of human fibronectin
1FBR 1995-08-08T00:00:00+0000 0 FOURTH AND FIFTH FIBRONECTIN TYPE I MODULE PAIR
1FNA 1994-01-11T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF THE TENTH TYPE III CELL ADHESION MODULE OF HUMAN FIBRONECTIN
1FNF 1995-09-30T00:00:00+0000 2.0 FRAGMENT OF HUMAN FIBRONECTIN ENCOMPASSING TYPE-III REPEATS 7 THROUGH 10
1FNH 1999-01-28T00:00:00+0000 2.8 CRYSTAL STRUCTURE OF HEPARIN AND INTEGRIN BINDING SEGMENT OF HUMAN FIBRONECTIN
1J8K 2001-05-22T00:00:00+0000 0 NMR STRUCTURE OF THE FIBRONECTIN EDA DOMAIN, NMR, 20 STRUCTURES
1O9A 2002-12-11T00:00:00+0000 0 Solution structure of the complex of 1F12F1 from fibronectin with B3 from FnBB from S. dysgalactiae
1OWW 2003-03-31T00:00:00+0000 0 Solution structure of the first type III module of human fibronectin determined by 1H, 15N NMR spectroscopy
1Q38 2003-07-28T00:00:00+0000 0 Anastellin

Relevant UniProtKB Entries

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