FIELD OF THE INVENTION

This relates to the field of proteins and, more particularly, to protein synthesis and design.

SEQUENCE LISTING

The application contains a Sequence Listing electronically submitted via EFS-web to the United States Patent and Trademark Office as a text file named “Sequence_Listing.txt,” which was created May 6, 2015 and is 3.08 kB. The electronically filed Sequence Listing serves as both the paper copy required by 37 C.F.R. § 1.821(c) and the computer readable file required by 37 C.F.R. § 1.821(c). The information contained in the Sequence Listing is incorporated by reference herein in its entirety.

BACKGROUND

Proteins are biomolecules made of amino acids linked together by peptide bonds to form amino acid sequences. Proteins perform a plethora of important functions in nature. Those functions are governed by their amino acid sequences and structure.

Proteins fold into complicated three-dimensional structures, which are characterized by four different structural terms. The primary structure of a protein is the linear representation of the protein's amino acid sequence. The secondary structure is the three-dimensional form of local segments of the protein, such as alpha-helices and beta-sheets. The tertiary structure is the protein's overall three-dimensional shape, fold, or architecture. The quaternary structure is the assembly of multiple polypeptide chains into a larger protein assembly, sometimes referred to as oligomeric assembly.

The tertiary structure forms through a process called “protein folding” in which some of the protein's amino acids interact with each other to cause the protein to fold into its three-dimensional conformation. Although the structure of a folded protein is complex, it is often symmetric to some degree. Therefore, in a symmetric protein, the tertiary structure can be simplified as a series of structural regions that appear multiple times in the protein.

Amino acid sequence segments that play a key role in folding a protein form what is called a “folding nucleus.” Studies have shown that the folding nucleus typically includes one-third to one-half of the overall polypeptide chain of single-domain globular proteins. Folding nuclei may be difficult to identify as they are not always defined by exon boundaries or contained neatly within an apparent structural repeating motif; they are considered to be a “cryptic” region within a protein. It appears that the presence of a folding nucleus is a protein design requirement, but there is no clear recipe for using a folding nucleus in protein design or how to complete the design of the remaining segments of the polypeptide to produce a robustly foldable protein.

BRIEF SUMMARY

This problem is overcome by designing proteins using folding peptide segments from a folding nucleus of a reference protein to form folding nuclei in synthetic proteins. The folding peptide segments are repeated throughout the synthetic protein's primary structure to form the remainder of the polypeptide sequence. This technique advantageously produces stable folding proteins.

A method of making a foldable protein embodying this design principle includes determining a folding nucleus peptide sequence associated with folding a reference foldable protein. The reference foldable protein has a tertiary structure. A synthetic foldable protein having a tertiary structure emulating the tertiary structure of the reference foldable protein is synthesized by including the determined folding nucleus peptide sequence and at least one repeat thereof in the peptide sequence of the synthetic foldable protein.

The step of determining the folding nucleus peptide sequence of the reference foldable protein may include identifying the folding nucleus of the reference foldable protein by performing “φ-value” analysis on the reference foldable protein.

The number of symmetry operations that can be performed on the reference foldable protein may be equal to a number of tandem repeats of the determined folding nucleus peptide sequence in the peptide sequence of the synthetic foldable protein.

In a particular embodiment, the synthetic foldable protein includes SEQ ID NO: 2.

In a particular embodiment, the reference foldable protein is native human FGF-1.

In a particular embodiment, the folding nucleus peptide sequence includes SEQ ID NO: 3.

Examples of tertiary structure that may be emulated include β-trefoil, TIM-barrel, or beta-propeller structures.

The tertiary structure of the synthetic foldable protein may have substantially the same symmetry as the tertiary structure of the reference foldable protein.

