Maize Codon Optimized Protein Expression in Pseudomonas fluorescens
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Solution Overview
Problem
Current Pseudomonas fluorescens expression systems face limitations in producing large quantities of recombinant proteins due to factors such as stem loop structures, exon:intron junctions, poly A addition signals, and restriction enzyme binding sites, which hinder protein expression and stability.
Innovation Solution
A codon optimization method is employed, specifically using Zea mays preferred codons to modify the polynucleotide sequence, removing detrimental elements like TA or CG doublets, [G+C] blocks, and restriction enzyme sites, resulting in enhanced protein expression by optimizing the coding sequence for Pseudomonas fluorescens host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional polynucleotide sequences are used for protein expression in Pseudomonas fluorescens, then the expression system can function, but protein production levels are limited due to detrimental sequence elements
Solution Approach 1:
The patent removes detrimental sequence elements (stem loop structures, exon:intron junctions, poly A addition signals, restriction enzyme binding sites) from the polynucleotide sequence through codon optimization, extracting only the necessary coding information while eliminating harmful components that limit protein production
Solution Approach 2:
The patent changes the codon usage parameters of the polynucleotide sequence to match Pseudomonas fluorescens preferences, optimizing translation efficiency. This involves altering the nucleotide sequence parameters while maintaining the same amino acid sequence, thereby improving protein production levels
2Productivity
If codon optimization is performed to increase protein expression, then recombinant protein production increases significantly, but the polynucleotide sequence must be carefully redesigned
Solution Approach 1:
The patent systematically changes codon parameters of the polynucleotide sequence to optimize for Pseudomonas fluorescens expression. By altering nucleotide choices at the codon level while maintaining amino acid sequences, the patent achieves high protein production through parameter optimization rather than complex structural redesign
Solution Approach 2:
The patent performs preliminary codon optimization of the polynucleotide sequence before introduction into the host cell. This preliminary redesign of the coding sequence ensures that the sequence is pre-optimized for the host's translation machinery, avoiding the need for complex post-introduction modifications
Data Source
AI summary
Compositions and methods for improving expression of a recombinant protein or polypeptide of interest in a host cell are provided. Compositions comprising a polynucleotide coding sequence for a BTBooster are provided using a plant optimized system. The coding sequences can be used in vector constructs or expression systems for transformation and expression of a recombinant protein or polypeptide of interest in a host cell. Methods comprising the codon optimization of a polynucleotide coding sequence for increased expression in a bacterial host are provided. The codon optimization method can be used in designing a polynucleotide coding sequence which expresses robust levels of protein in a bacterial host cell.