GG Repeat Fusion Tags for Protein Renaturation and Proteolysis Resistance
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Solution Overview
Problem
Existing methods for recombinant protein production using the type 1 secretion system (T1SS) face challenges such as suboptimal solubility and susceptibility to proteolysis, limiting high-yield production of peptides and proteins.
Innovation Solution
The use of artificially combined GG repeat sequences, specifically in the form of GGR1-Linker-(GGRN-Linker)n-GGRC, as a fusion tag to enhance renaturation efficiency, stability, and solubility of recombinantly expressed peptides and proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If fusion proteins are produced using the type 1 secretion system (T1SS) with existing methods, then production capability is achieved, but solubility is suboptimal and susceptibility to proteolysis increases
Solution Approach 1:
The patent introduces an artificial GG repeat sequence as an intermediary fusion tag between the target peptide/protein and the secretion system. This GG repeat tag acts as a mediator that improves solubility of the fusion protein and protects against proteolytic degradation, while being compatible with the T1SS secretion mechanism. The tag can be subsequently removed after secretion to obtain the pure target molecule.
Solution Approach 2:
The fusion protein construct combines multiple functional elements: the target peptide/protein sequence, the artificial GG repeat sequence (consisting of multiple glycine-rich repeats), and the secretion signal sequence. This composite structure leverages the solubility-enhancing properties of glycine-rich sequences and the protective effects against proteolysis, while maintaining compatibility with the bacterial secretion system.
2Productivity
If existing fusion constructs are used for recombinant protein production, then expression is achieved, but high yields are prevented by suboptimal solubility
Solution Approach 1:
The patent modifies the amino acid sequence parameters by incorporating multiple glycine-rich repeat sequences with specific consensus patterns (e.g., GGxGxDxUx where U is hydrophobic). These sequence parameter changes fundamentally alter the physicochemical properties of the fusion protein, dramatically improving solubility and enabling high-yield production. The specific spacing and arrangement of glycine residues create favorable solvation characteristics.
3Productivity
If existing fusion constructs are used, then production is achieved, but high yields are prevented by susceptibility to proteolysis
Solution Approach 1:
The artificial GG repeat sequence serves as a protective cushion that precedes and shields the target peptide/protein from proteolytic attack. The glycine-rich repeats create a steric and structural barrier that protects the fusion protein from degradation by proteases in the periplasmic space and extracellular environment, thereby maintaining stability throughout the secretion and storage process.
Data Source
AI summary
The present invention relates to polypeptides comprising a first amino acid sequence comprising one or more GG repeat sequences and a peptide or polypeptide of interest in form of a fusion protein that exhibits increased renaturation efficiency and optionally also improved expression. Also encompassed are nucleic acids encoding these polypeptides, host cells that comprise said nucleic acids, and methods for protein expression and renaturation using said nucleic acids, host cells and polypeptides.