Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain - PubMed (original) (raw)

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

C Anders Olson et al. Protein Sci. 2007 Mar.

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.

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Figures

Figure 1.

Figure 1.

Sequence and structure of 10FnIII. (A) Sequence of 10FnIII. Residues that are part of the β-strand framework are in bold. Residue positions that were selected for randomization are colored red. (B) Two structural views of 10FnIII. The crystal structure of the 10FnIII domain shown is part of the 7–10FnIII domains (left, PDB accession no. 1FNF). The randomized BC and FG loop regions are colored red. The first seven N-terminal residues are colored green. The solution structure of 10FnIII is illustrated in a side view without the unstructured N-terminal residues (right, PDB accession no. 1TTG).

Figure 2.

Figure 2.

Purification and stability of WT 10FnIII compared to 10FnIII(Δ1–7). (A) Purification of WT 10FnIII (lanes 1–5) and 10FnIII(Δ1–7) (lanes 6–10). (Lanes 1,6) Protein standard. (Lanes 2,7) Cleared lysate. (Lanes 3,8) HisTrap column purification. (Lanes 4,9) Factor Xa cleavage of His6-tag. (Lanes 5,10) Gel filtration purification. (B) Guanidinium chloride denaturation of WT 10FnIII (open diamonds) and 10FnIII(Δ1–7) (closed squares) monitored by Trp fluorescence.

Figure 3.

Figure 3.

Scheme for constructing 10FnIII library. Linear illustration of library DNA (A) generated from eight oligonucleotides (B).

Figure 4.

Figure 4.

Random sequence composition. The frequencies of each amino acid (closed diamonds) are compared to the target NNS distribution (open circles) and the composition of natural protein interfaces involved in protein–protein binding (Chakrabarti and Janin 2002).

Figure 5.

Figure 5.

Expression analysis of 10FnIII library. (A) Expression fitness landscape of the 10FnIII library. Fluorescence intensity values of 94 Fn variant–GFP fusions were obtained from cell suspensions and normalized to WT 10FnIII(Δ1–7)-GFP. Each variant is marked by a dash on the X axis. (B) Relation between relative fluorescence and Fn variant soluble expression. Nineteen variants plus WT 10FnIII(Δ1–7) were subcloned into an expression vector without GFP. The relative expression values represent the amount of protein expressed in the soluble fraction relative to wild type measured by band densitometry. (C) The relation between relative fluorescence and Fn variant solubility, determined by band densitometry of soluble and insoluble fractions.

Figure 6.

Figure 6.

Guanidinium chloride denaturation of 10FnIII variants compared to WT10 FnIII(Δ1–7). (Closed triangles) Fn04; (open circles) Fn23; (open squares) Fn32; (closed circles) Fn38; (crosses) WT 10FnIII(Δ1–7).

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