Enterokinase-Cleavable Polypeptides for Specific Fusion Protein Removal
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Solution Overview
Problem
Current methods for removing fusion partner proteins using enterokinase suffer from limited substrate specificity, leading to unintended cleavage of internal sites in mature proteins and potential chemical or physical changes, necessitating a more specific and stable enterokinase cleavage reaction suitable for industrial protein production.
Innovation Solution
The development of Enterokinase-cleavable fusion polypeptides with a specific cleavage site (Z2-X6-X5-X4-G-D-R-Z1) that is hydrolyzed faster than secondary sites, ensuring chemical stability and efficient cleavage under mild conditions, where Z1 is a target polypeptide, X4 is E, Q, L, D, G, A, S, F, H, Y, W, or M, and X5 is selected from genetically encoded amino acids excluding S and I, facilitating the isolation of the target polypeptide.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If enterokinase is used for removing fusion partner proteins, then the fusion partner protein can be cleaved off, but enterokinase has limited substrate specificity and may cleave internal sites in the mature protein
Solution Approach 1:
The patent modifies the local amino acid sequence at the cleavage site to have unique properties that distinguish it from other sites. By introducing specific amino acids (such as proline or cysteine) at predetermined positions in the D4K sequence, the cleavage site acquires local characteristics that enhance enterokinase recognition and cleavage efficiency while preventing cleavage at other sites in the mature protein.
Solution Approach 2:
The patent changes the chemical parameters of the cleavage site by substituting specific amino acids at defined positions. For example, replacing certain residues with proline (which has a rigid cyclic structure) or cysteine (which can form disulfide bonds) alters the local conformation and chemical properties, making the cleavage site more specific and resistant to unintended cleavage while maintaining efficiency.
2Productivity
If enterokinase cleavage is performed under standard conditions, then cleavage can occur, but unintended chemical or physical changes may occur to the mature protein
Solution Approach 1:
The patent modifies the cleavage site sequence to enable efficient cleavage under milder conditions. By introducing specific amino acid substitutions (such as adding basic residues like arginine or lysine near the cleavage site), the local positive charge enhances interaction with enterokinase, allowing cleavage to proceed efficiently at lower temperatures and milder pH conditions that preserve the stability of the mature protein.
Solution Approach 2:
The modified cleavage site acts as an intermediary that facilitates the interaction between enterokinase and the fusion protein. The introduced amino acids (such as proline or cysteine) create a unique structural motif that serves as a specific recognition element, enabling the enzyme to distinguish the intended cleavage site from other potential sites and to cleave efficiently without requiring harsh conditions that could damage the mature protein.
3Quantity of substance
If the D4K processing site is used for enterokinase cleavage, then the fusion partner can be removed, but the hydrolysis rate at the D4K site may be slower than at internal degradation sites
Solution Approach 1:
The patent enhances the local quality of the D4K cleavage site by introducing specific amino acids at predetermined positions. For example, adding proline at position -2 or cysteine at position -3 creates a unique local structure that is recognized more efficiently by enterokinase, increasing the hydrolysis rate at this specific site while maintaining selectivity against internal sites.
Solution Approach 2:
The patent changes the chemical parameters of the D4K site by substituting amino acids to introduce favorable chemical groups. For instance, adding basic residues (arginine, lysine) near the cleavage site increases local positive charge, which enhances electrostatic interaction with the enzyme's active site, thereby accelerating cleavage at the D4K site relative to internal sites.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach ensures precise cleavage of fusion partner proteins without affecting the mature protein, maintaining chemical stability and preventing unwanted modifications, making it suitable for industrial-scale protein production.
Implementation Method 1
The protein of interest is often attached to a fusion partner protein or a smaller amino acid extension in order to increase the expression level, facilitate secretion, increase the solubility, promote protein folding, to protect the protein against unintentional proteolysis or to facilitate purification of the protein of interest. The fusion partner protein needs to be removed from the fusion protein by proteolysis to obtain the protein of interest.
Implementation Method 2
One protease used for such processing is enterokinase (E.C. 3.4.21.9). The biologically natural function of this protease is to convert trypsinogen into trypsin by cleavage at a DDDDK processing site (SEQ ID NO: 2, hereafter D4K) in the zymogen
Data Source
AI summary
The present invention relates to Enterokinase-cleavable polypeptides comprising an Enterokinase cleavage site connected to a polypeptide and their use for making the target polypeptide by expression. The invention also relates to DNA sequences, vectors and host cells for use in expressing the Enterokinase-cleavable polypeptides.