Plant Expression Vectors With Double Terminators for Higher Protein Yield
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Solution Overview
Problem
Existing plant-based recombinant protein production systems have not systematically optimized gene terminators and surrounding regions, limiting protein expression efficiency.
Innovation Solution
Incorporation of optimized 3′ UTRs, including double terminators and chromatin scaffold/matrix attachment regions (MARs) in plant expression vectors, enhancing protein expression by increasing transcript stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional terminators are used in plant expression vectors, then the system is simple and easy to manufacture, but protein expression levels are limited
Solution Approach 1:
The patent combines multiple terminator sequences (first terminator and second terminator) into a single 3' UTR region of the expression cassette. This merging of functional elements creates a composite terminator structure that synergistically enhances transcription termination efficiency and protein expression levels, resolving the contradiction between simplicity and productivity.
Solution Approach 2:
The 3' UTR is constructed as a composite element incorporating multiple functional sequences including terminators, MARs, and other regulatory motifs. This composite structure integrates diverse functional components to achieve enhanced transcriptional control and protein production, transforming a simple terminator into a multi-functional expression enhancement module.
2Productivity
If optimized 3' UTRs with double terminators and MARs are incorporated, then protein expression increases significantly, but the vector construction complexity increases
Solution Approach 1:
The 3' UTR is segmented into distinct functional modules: first terminator, second terminator, and MAR regions. This segmentation allows independent optimization and modular assembly of each component, facilitating easier vector construction while maintaining the synergistic effects that drive high protein expression.
Solution Approach 2:
The MAR (matrix attachment region) acts as an intermediary element between the terminators and the chromatin structure. It mediates the interaction between the terminator sequences and nuclear machinery, enhancing transcription termination efficiency without requiring direct complex interactions between all components, thus simplifying the overall system design.
Data Source
AI summary
The present disclosure relates to plant-based recombinant protein production systems and their methods of production and use. The plant-based recombinant protein production system is a vector comprising a 5′ UTR and a 3′ UTR, wherein 3′ UTR comprises at least one terminator selected from the group consisting of: EU, IEU, NbACT3, NbACT617, NbACT567, Pin2, BDB501, BDB282, NbHSP, NbHSPb, Rep, RbcS, SIR, SIR 5′/3′, SIR 3′, AtHSP, 35S, RepA, NOS, TMV, TNVD, PEMV, and BYDV. In certain implementations, the vector comprises two terminators in 3′ UTR, where the two terminators are fused to form a double terminator. For example, the double terminator comprises two members selected from the group consisting of: EU, IEU, NbACT3, NbACT617, NbACT567, Pin2, BDB501, BDB282, NbHSP, NbHSPb, Rep, RbcS, SIR, SIR 5′/3′, SIR 3′, AtHSP, 35S, RepA, NOS, TMV, TNVD, PEMV, and BYDV. In some aspects, the vector further comprises a chromatin scaffold/matrix attachment region (MAR) that is downstream of the terminators.