Plant Protein Expression Vectors with Composite Promoters and Terminators
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Solution Overview
Problem
Current methods for producing recombinant proteins in plants face challenges due to low gene expression levels, limiting their competitiveness compared to other systems, and there is a lack of techniques for combining strong promoter sites from plant viruses and repeatedly linking terminators to enhance expression.
Innovation Solution
A gene construct is developed, incorporating a super promoter (FMM-UD) formed by linking Figwort and Mirabilis mosaic virus promoter fragments and a GAL4-binding site, and a strong terminator (3PR) created by connecting cauliflower mosaic virus and potato proteinase inhibitor II gene regions, to increase protein expression in plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional promoters and terminators are used in plant expression systems, then the system is simple and easy to construct, but the gene expression level remains low, limiting protein production efficiency
Solution Approach 1:
The patent combines multiple promoter elements (CaMV 35S promoter, Ocs promoter, and viral promoter fragments) to create a hybrid super-promoter that integrates the strengths of each individual promoter, achieving synergistic enhancement of transcription initiation and elongation efficiency. Similarly, multiple terminator sequences are linked to create a composite terminator structure that improves transcription termination and mRNA stability.
Solution Approach 2:
The expression system uses composite regulatory elements formed by linking multiple functional DNA sequences together. The super-promoter comprises composite structures including TATA boxes, GC-rich regions, and viral promoter sequences. The enhanced terminator consists of composite sequences from different sources (CaMV 35S terminator, nopaline synthase terminator fragments) that work together to improve transcription termination efficiency.
2Productivity
If strong promoters from plant viruses are recombined to create a super promoter, then transcription level and protein expression increase significantly, but the construction complexity of the gene construct increases
Solution Approach 1:
The super-promoter is constructed by segmenting different functional elements from various viral and plant promoters into discrete modules. Each module (e.g., TATA box region, GC-rich region, viral promoter fragment) is independently optimized and then assembled in a specific arrangement to achieve cumulative transcriptional activation effects while maintaining modular flexibility for future modifications.
3Productivity
If terminators are repeatedly linked to create a strong terminator, then transcription termination efficiency and gene expression improve, but the length and complexity of the gene construct increase
Solution Approach 1:
The patent applies partial repetition of terminator sequences rather than exhaustive linkage of all possible terminator elements. By selecting and repeating only the most effective terminal sequences (such as linking CaMV 35S terminator with nopaline synthase terminator fragments), the system achieves sufficient transcription termination efficiency without unnecessarily extending the overall construct length beyond what is functionalally required.
Data Source
AI summary
The present invention relates to a high expression vector, including a strong recombinant promoter and a terminator, for mass-producing a protein of interest in a plant, and a method for mass-producing a protein of interest using the same. More specifically, the present invention provides a method for mass-producing a protein of interest in a plant by increasing the expression of the protein of interest by increasing the transcription level of the protein of interest by using a super promoter, produced by recombining strong promoter sites derived from plant viruses, and/or a strong terminator, produced by repeatedly connecting two types of terminators.