Bacterial Expression of Water-Soluble Moesin-Binding Polypeptides
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Solution Overview
Problem
Existing methods for forming recombinant proteins in bacterial expression systems are limited by the formation of cytoplasmic aggregates (inclusion bodies) that are water insoluble, and eukaryotic systems offer lower yields and are more complicated.
Innovation Solution
A method to produce water-soluble polypeptides by altering insoluble proteins using bacterial expression systems, involving solubilization with urea and biotinylation, and using moesin binding partners for detection and isolation of naïve NK cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant proteins are produced in bacterial expression systems, then high-level expression is achieved, but the proteins form water-insoluble cytoplasmic aggregates (inclusion bodies)
Solution Approach 1:
The patent applies parameter changes by modifying the solubility characteristics of the recombinant protein through amino acid substitutions. Specifically, hydrophobic residues are replaced with hydrophilic residues in the protein sequence, which fundamentally changes the protein's interaction with water and its aggregation propensity, thereby maintaining solubility at high expression levels in bacterial systems
Solution Approach 2:
The patent uses solubility-enhancing tags or fusion proteins as intermediaries that temporarily associate with the recombinant protein during expression. These intermediaries act as mediators that prevent aggregation of the target protein while maintaining its structural integrity, and can be removed later through proteolytic cleavage or affinity purification
2Reliability
If eukaryotic expression systems are used to produce water-soluble proteins, then solubility is improved, but yields are lower and the system becomes more complicated
Solution Approach 1:
The patent fundamentally changes the solubility parameter of the protein by introducing amino acid substitutions that increase hydrophilicity. This parameter change allows the protein to maintain water solubility when expressed in simple bacterial systems, thereby achieving both high solubility and high productivity without requiring complex eukaryotic expression machinery
Solution Approach 2:
The patent extracts or removes the problematic hydrophobic regions from the protein sequence that cause aggregation. By taking out or replacing these hydrophobic segments with hydrophilic alternatives, the protein maintains its functional domains while eliminating the aggregation-prone regions, enabling high-yield expression in bacterial systems
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
Enables high-level expression and recovery of functionally active water-soluble proteins, facilitating efficient detection, counting, and isolation of naïve NK cells in biological samples.
Implementation Method 1
contacting a polypeptide, fragment, variant, or fusion polypeptide precipitate obtained from bacteria cells with an aqueous urea solution in an amount sufficient to solubilize the precipitate and form a dissolved polypeptide, fragment, variant, or fusion polypeptide within an aqueous medium
Implementation Method 2
contacting the dissolved polypeptide, fragment, variant, or fusion polypeptide within the aqueous medium with biotin under conditions sufficient to bind biotin to the dissolved polypeptide, fragment, variant, or fusion polypeptide and form biotinylated dissolved polypeptide, fragment, variant, or fusion polypeptide
Data Source
AI summary
The present disclosure relates to a polypeptide which is a variant of SEQ ID NO:1, fragment thereof, or fusion polypeptide, including: the variant of SEQ ID NO:1 or a fragment thereof, having an increased water solubility compared to SEQ ID NO:1 which binds to moesin. In embodiments, the polypeptide includes an affinity tag. Compositions and methods of detecting naïve NK cells using one or more moesin binding partners are also disclosed.