Hepatitis B Cis-Regulatory Element for Protein Expression
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Solution Overview
Problem
Current methods for protein expression in eukaryotic cells, such as those using commercial vectors like pGL4.10 and pTargeT, face challenges in achieving high efficiency and specificity, particularly in cell-free systems and cells of various origins, necessitating the development of enhanced regulatory sequences for exogenous protein expression.
Innovation Solution
The use of cis-acting regulatory elements derived from the preS1 region of the Hepatitis B virus with specific mutations, which function to enhance protein expression by acting as a promoter in eukaryotic cells, increasing translational efficiency and protein production levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If commercial vectors with conventional promoters and enhancers are used for protein expression in eukaryotic cells, then basic protein expression is achieved, but high efficiency and specificity are not attained
Solution Approach 1:
The patent applies parameter changes by modifying the nucleotide sequence of the cis-acting element to create an optimized version with enhanced translational efficiency. The specific parameter changed is the nucleotide sequence composition, which was altered to improve ribosome binding and translation initiation, thereby resolving the contradiction between expression efficiency and specificity.
Solution Approach 2:
The patent applies local quality by introducing specific mutations at critical positions within the cis-acting element sequence, particularly around the start codon region. These localized sequence modifications enhance translational efficiency without affecting the overall structure or function of the expression system, allowing high efficiency and specificity to coexist.
2Productivity
If existing regulatory sequences are used to enhance exogenous protein expression, then some expression improvement is achieved, but sufficient enhancement across various cell origins and characteristics is not obtained
Solution Approach 1:
The patent applies universality by designing a cis-acting element with conserved functional features that can operate effectively across different eukaryotic cell types and origins. The optimized sequence maintains compatibility with various promoters and expression systems, making it universally applicable while achieving high expression levels, thus resolving the contradiction between productivity and adaptability.
3Productivity
If conventional expression systems are used, then standard protein production is achieved, but high translational efficiency without affecting transcriptional levels is not realized
Solution Approach 1:
The patent applies segmentation by separating the control of transcriptional and translational processes. The cis-acting element is designed to specifically influence translation initiation and efficiency without interfering with transcriptional regulation. This segmentation allows independent optimization of translational efficiency while maintaining precise control over transcriptional levels, resolving the contradiction between productivity and manufacturing precision.
Data Source
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AI summary
The present invention provides a eukaryotic cis-regulatory element selected from the group consisting of polynucleotides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. The present invention provides the cis acting element and an expression vector for animal cells containing an operable promoter in eukaryotic cells. The vector of the present invention allows a recombinant protein to be effectively expressed in animal cells. The vector of the present invention and the method for manufacturing the recombinant protein using same can be effectively applied in the industrial mass production of medicines and medical supplies.