TIS and Signal Peptide Sequence for Predictable Protein Expression
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Solution Overview
Problem
Existing recombinant expression systems in mammalian cells, such as CHO cells, face unpredictable and context-dependent variations in protein expression levels, limiting the rational design for achieving desired translation initiation rates and protein yields.
Innovation Solution
A novel TIS sequence, TISEVO (TCGGTCATGGC), is introduced to enhance translation initiation rates by optimizing the nucleotide sequence surrounding the ATG start codon, maintaining protein quality and biosimilarity, and promoting efficient ribosome recruitment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the conventional GCCACCATG TIS sequence is used, then translation initiation occurs, but protein expression levels vary unpredictably and context-dependently
Solution Approach 1:
The patent modifies the TIS sequence parameters by replacing the conventional GCCACCATG sequence with the optimized TCGGTCATGGC sequence. This parameter change in the nucleotide composition and arrangement directly addresses the unpredictability of protein expression levels while maintaining translation initiation functionality.
2Productivity
If translation initiation rate is increased to boost protein yield, then productivity improves, but protein quality and post-translational modifications may be affected
Solution Approach 1:
The optimized TCGGTCATGGC TIS sequence is designed to enhance translation initiation rates while preserving the reading frame and start codon integrity. This parameter optimization increases productivity without compromising protein quality or post-translational modifications, as the core translational machinery remains intact.
Data Source
AI summary
The present invention relates to DNA constructs comprising a novel TIS sequence. The TIS sequence transcribes into an RNA motif that functions as the protein translation initiation site in an mRNA transcript. The DNA construct may further comprise a nucleic acid sequence encoding signal peptide. Additionally, the DNA constructs may also comprise a nucleic acid sequence encoding a recombinant protein or one or more polypeptide chains thereof. The invention further relates to an expression vector, expression cassette and host cell which comprise said DNA constructs. Furthermore, the present invention relates to a recombinant protein expressed by the host cell as well as a method for expressing the recombinant protein.