Periplasmic Targeting Signal Sequences for Soluble Protein Production
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Solution Overview
Problem
The production of biologically active recombinant proteins in bacterial hosts often results in insoluble aggregates known as inclusion bodies, which complicates the cost-effective production of therapeutic proteins and requires additional steps for purification and refolding, and existing methods for improving folding, such as using fusion proteins or molecular chaperones, have limited effectiveness.
Innovation Solution
A method involving the expression of a modified polypeptide with an accessory polypeptide linked to the biologically active polypeptide, which increases the yield of soluble, active protein, with the accessory polypeptide enhancing solubility and serum half-life, and can be expressed in various host cells including prokaryotes, eukaryotes, and mammalian cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant proteins are expressed in the cytoplasm of E. coli, then production yield is improved, but the proteins form insoluble aggregates known as inclusion bodies
Solution Approach 1:
The patent introduces periplasmic targeting signal sequences as intermediary elements that redirect protein expression from the cytoplasmic environment (where aggregates form) to the periplasmic space (where proteins remain soluble). This mediator approach resolves the contradiction by changing the localization environment without altering the protein sequence itself.
Solution Approach 2:
The patent changes the environmental parameter of protein expression location from cytoplasm to periplasm by adding signal sequences. This parameter change transforms the physical-chemical environment where proteins fold and assemble, preventing hydrophobic aggregation while maintaining high production yields.
2Reliability
If inclusion bodies are purified and refolded to obtain soluble protein, then active protein is obtained, but additional processing steps and time are required
Solution Approach 1:
The patent applies preliminary action by pre-equipping the expression system with periplasmic targeting signal sequences before protein expression. This preliminary modification ensures that proteins are directly targeted to the periplasm where they fold correctly and remain soluble, eliminating the need for subsequent refolding steps and reducing processing time.
Solution Approach 2:
The patent extracts the problematic refolding step from the production process by preventing aggregate formation in the first place through periplasmic targeting. This removes the time-consuming denaturation and refolding operations while still obtaining active, soluble protein.
3Reliability
If fusion proteins or molecular chaperones are used to improve folding, then solubility is enhanced, but the methods have limited effectiveness and increased complexity
Solution Approach 1:
The patent uses periplasmic targeting signal sequences as a simple intermediary element that naturally mediates proper protein folding by directing proteins to the periplasmic environment. This approach is more effective and simpler than fusion proteins or molecular chaperones, as it relies on the native folding capabilities of the periplasm without requiring additional complex components.
Data Source
AI summary
The present invention relates to biologically active polypeptides linked to one or more accessory polypeptides. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applications.