Fusion Tags Enhance rhPTH Solubility and Yield in E. coli
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current protein expression systems, particularly in E. coli, face challenges such as low expression levels, protein insolubility, and improper folding when producing recombinant human parathyroid hormone (rhPTH), which limits therapeutic applications for osteoporosis treatment.
Innovation Solution
Development of novel fusion tags of 20-40 amino acids, such as DSLRFLYDGIRIQADQAPEDLDMELGS, and their use in expression vector systems with a His-tag and TEV cleavage site, allowing for high-level expression and subsequent purification of rhPTH(1-34) in E. coli.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If small peptides such as rhPTH are expressed in E. coli, then production of therapeutic proteins is achieved, but expression levels are low
Solution Approach 1:
The patent uses fusion tags (GST, Trx, His-tag) as intermediary proteins that facilitate the expression and purification of rhPTH. These tags act as mediators that enhance solubility and enable efficient purification through affinity chromatography, thereby improving both expression levels and yield of the target peptide.
Solution Approach 2:
The patent creates composite fusion proteins by combining rhPTH with fusion tags (GST-rhPTH, Trx-rhPTH, His-tag-rhPTH). These composite structures leverage the beneficial properties of each component: the solubility and stability of the fusion tag combined with the therapeutic function of rhPTH, resulting in improved expression and yield.
2Stability of the object's composition
If fusion tags are used to improve solubility of expressed proteins, then solubility is enhanced, but final yield is compromised due to larger size of fusion partner
Solution Approach 1:
The patent segments the fusion protein into distinct functional domains: the fusion tag portion and the rhPTH portion. By introducing specific cleavage sites (TEV protease site, Thrombin site) between these segments, the system allows for easy separation after expression, enabling recovery of pure rhPTH while benefiting from the solubility enhancement during the expression phase.
Solution Approach 2:
The patent optimizes the size and composition parameters of fusion tags to balance solubility enhancement with minimal impact on yield. The His-tag (6-10 histidine residues) and other fusion partners are carefully designed to provide sufficient solubility improvement while maintaining manageable molecular weights that do not excessively reduce the final yield of the target protein.
3Stability of the object's composition
If available fusion expression systems (GST, Trx, Sumo) are used, then proteins become soluble, but expression efficiency is reduced
Solution Approach 1:
The patent employs fusion tags that serve multiple functions simultaneously: GST and Trx provide solubility enhancement, His-tag enables affinity purification and detection, and the fusion construct itself promotes proper folding. This multi-functionality increases expression efficiency by addressing multiple challenges (solubility, purification, stability) with a single fusion partner design.
Solution Approach 2:
The fusion tags act as intermediaries that facilitate various stages of protein production: they mediate solubility during expression, mediate purification through affinity chromatography, and mediate proper folding. This intermediary function across multiple process stages enhances overall expression efficiency compared to using no fusion tag or single-function tags.
Data Source
AI summary
The present invention provides for fusion tags of 20 to 40 amino acids having a sequence as given in FIG. 1, fusion tags sharing homology with these fusion tags, or hybrid fusion tags comprising sequences of FIG. 1. The invention further provides an expression vector system comprising a fusion tag of the invention, and a process of obtaining recombinant human parathyroid hormone by using the fusion tags and the expression vector system of the invention.