HlyA Fusion Variants for Soluble and Stable Protein Secretion
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Solution Overview
Problem
Existing methods for recombinant protein production using Type 1 secretion systems, particularly those involving HlyA, face issues with suboptimal solubility and susceptibility to proteolysis, leading to reduced yields and stability of fusion constructs.
Innovation Solution
Development of HlyA variants with specific amino acid substitutions or deletions at defined positions, enhancing stability and solubility, and increasing expression rates of fusion proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If HlyA or HlyA1 is used as fusion partner for recombinant protein production, then peptide and protein expression can be achieved through Type 1 secretion system, but the fusion constructs exhibit suboptimal solubility and susceptibility to proteolysis
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (161, 162, 163, 165, 171, 176, 179, 180, 181, 186, 187, 188, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 205, 206, 209, 210, 212, 218) of HlyA1 to generate variants with improved solubility and proteolytic stability. Over 400 variants were created through substitutions and deletions, with specific examples including variants lacking residues 191-194, and variants with substitutions at positions 162, 165, 171, 179, 188, 190, 191, 193, 194, 198, 199, 201, 205, 209, 212, and 218
Solution Approach 2:
The patent applies the extraction principle by removing specific amino acid sequences from HlyA1 that are responsible for proteolytic susceptibility. Notable examples include deletion variants lacking residues 191-194 (QKPA), 198-201 (KPEK), or 209-218 (GQGQGQGQ), and deletion of the C-terminal region. These extracted modifications eliminate protease recognition sites while preserving the secretion and solubility functions
2Reliability
If HlyA variants with amino acid substitutions or deletions are introduced, then solubility and stability are improved, but expression rates may be affected
Solution Approach 1:
The patent systematically explores parameter changes by testing numerous amino acid substitutions and deletions to identify modifications that improve solubility and stability without compromising expression rates. The screening process evaluated over 400 variants to find the optimal balance between stability improvement and expression maintenance
Solution Approach 2:
The patent employs feedback mechanisms by screening and characterizing multiple HlyA variants to identify those that simultaneously achieve improved solubility/stability and maintain acceptable expression rates. The iterative process of creating variants, testing their properties, and selecting the best performers allows optimization of both parameters
Data Source
AI summary
Polypeptides comprising hemolysin A (Hly A) fragments and/or variants and a peptide or polypeptide of interest in form of a fusion protein that exhibits improved expression and secretion as well as increased solubility and stability and improved renaturation efficiency. Also encompassed are nucleic acids encoding these polypeptides. host cells that comprise said nucleic acids, and methods and uses for protein expression using said nucleic acids, host cells and polypeptides.