GG Repeat Fusion Tags for Recombinant Protein Solubility
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Solution Overview
Problem
Current methods for recombinant protein production using the type 1 secretion system face challenges with suboptimal solubility and susceptibility to proteolysis, leading to reduced yields and stability of fusion constructs.
Innovation Solution
Employing specific amino acid sequences derived from RTX proteins, featuring GG repeat motifs, as fusion tags to enhance the renaturation efficiency and stability of recombinantly expressed peptides and proteins, allowing for higher yields and protection against degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If fusion proteins are used for recombinant protein production, then expression rates are improved, but solubility and stability deteriorate due to suboptimal solubility and susceptibility to proteolysis
Solution Approach 1:
The patent introduces an intermediary element - a specific amino acid sequence derived from RTX proteins with GG repeat motifs - that acts as a mediator between the fusion protein and the host cell secretion system. This intermediary sequence improves solubility and stability while maintaining expression rates, resolving the contradiction between productivity and reliability
Solution Approach 2:
The patent applies parameter changes by modifying the amino acid sequence composition - specifically incorporating GG repeat motifs (GGxGxDxUx) at defined positions within the fusion protein. These compositional changes alter the physical-chemical properties of the protein, improving solubility and resistance to proteolysis while maintaining expression efficiency
2Reliability
If longer RTX-derived sequences are used, then solubility and stability are improved, but expression rates decrease
Solution Approach 1:
The patent identifies optimal parameter ranges for the RTX-derived sequence length (30-200 amino acids) and GG repeat positioning. By changing these parameters to specific optimal values, the patent achieves both improved solubility/stability and maintained high expression rates, resolving the trade-off between reliability and productivity
3Productivity
If fusion constructs are produced, then production yield is improved, but susceptibility to proteolysis increases
Solution Approach 1:
The RTX-derived amino acid sequence acts as a protective intermediary that shields the fusion construct from proteolytic degradation. This intermediary sequence with GG repeats serves as a protective layer that maintains production yield while reducing susceptibility to proteolysis, degradation, and other harmful factors
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.