Batroxobin Codon Optimization for Yeast Expression Yield
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Solution Overview
Problem
Current methods for producing recombinant thrombin-like enzymes, such as batroxobin, in E. coli result in low yields and inactive proteins due to gene codon mismatch issues, with no reported cases of recombinant thrombin-like enzymes achieving similar activity to natural enzymes.
Innovation Solution
Development of a recombinant expression system using mutated batroxobin cDNAs and α-factor secretion signal sequences in yeast, specifically Pichia pastoris, to enhance protein translation efficiency and secretion, resulting in higher yields and biologically active batroxobin production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant batroxobin is produced in E. coli using native sequences, then production is attempted, but yield is low and protein is inactive due to gene codon mismatch issues
Solution Approach 1:
The patent applies codon optimization by changing the nucleotide sequence parameters of batroxobin cDNA to match E. coli preferred codon usage patterns. This involves substituting synonymous codons throughout the gene sequence to improve translation efficiency while maintaining the same amino acid sequence, thereby resolving the codon mismatch issue that caused low yield and inactivity
Solution Approach 2:
The patent creates an optimized copy of the batroxobin gene with modified nucleotide sequences that are better adapted to E. coli expression systems. This copied version maintains the functional amino acid sequence while having improved codon composition for prokaryotic translation machinery
2Productivity
If recombinant batroxobin is produced in E. coli, then production process is implemented, but protein secretion and translation efficiency are insufficient
Solution Approach 1:
The patent modifies multiple parameters of the expression system including codon usage frequency, GC content, and addition of prokaryotic promoter elements and ribosome binding sites to the batroxobin gene construct, thereby optimizing translation efficiency for E. coli without significantly increasing process complexity
Solution Approach 2:
The patent performs preliminary optimization of the gene sequence before expression, including codon pre-adaptation to E. coli preferences and inclusion of necessary regulatory elements, so that the gene is ready for efficient expression without requiring complex post-expression processing
3Productivity
If native batroxobin sequences are used in recombinant expression, then natural protein structure is maintained, but expression yield is low
Solution Approach 1:
The patent changes nucleotide-level parameters (codon selection, GC content) while maintaining the amino acid sequence fidelity, achieving higher expression yield without altering the protein's primary structure or functional properties
Solution Approach 2:
The patent creates a nucleotide sequence copy that is functionally equivalent but optimized for expression, where the DNA/RNA sequence differs but the encoded protein sequence remains identical to the native batroxobin
Data Source
AI summary
The present invention relates to a batroxobin-encoding nucleotide sequence and/or a mutated α-factor secretion signal sequence, and a vector and a transformant using the same. The batroxobin-encoding nucleotide sequence of this invention exhibits an excellent expression efficiency in yeast, particular Pichia pastoris and the recombinant batroxobin is obtained at 4-13 fold higher yield than natural-occurring batroxobin-encoding sequences. The protein expression system which uses the batroxobin-encoding nucleotide sequence as well as mutated α-factor secretion signal peptide sequence of this invention obtains the recombinant batroxobin at about 20-fold higher yield than natural-occurring batroxobin-encoding sequences. In addition, the recombinant batroxobin prepared using the sequence of this invention has a significantly plausible activity and stability compared with natural-occurring batroxobin.