Optimized rhBMP-2 Sequence for High-Yield Prokaryotic Expression
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Solution Overview
Problem
Current methods for producing recombinant human bone morphogenetic protein-2 (rhBMP-2) face challenges such as low expression levels, high production costs, and poor renaturation and purification effects, leading to reduced biological activity and availability, particularly in prokaryotic expression systems which lack post-translational modifications.
Innovation Solution
Optimization of the nucleotide sequence of rhBMP-2 to create a novel amino acid sequence that is more suitable for expression in E. coli, including modifications such as adding methionine at the N-terminal and using E. coli-preferred codons, followed by renaturation and multi-step ion exchange chromatography for purification, to enhance stability and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If prokaryotic expression system is used to produce rhBMP-2, then production cost is reduced and expression level can be increased, but biological activity and stability deteriorate due to lack of post-translational modifications
Solution Approach 1:
The patent optimizes the nucleotide sequence parameters of the rhBMP-2 gene by using E. coli-preferred codons and removing rare codons, which changes the translation efficiency and protein expression level while maintaining biological activity through optimized coding sequence rather than changing the protein structure itself
2Reliability
If eukaryotic expression system is used to produce rhBMP-2, then biological activity is improved through proper folding and post-translational modification, but production cost increases and expression level decreases
Solution Approach 1:
The patent creates a simplified copy of the eukaryotic expression system by using prokaryotic E. coli cells with an optimized nucleotide sequence that mimics the functional characteristics needed for high biological activity, thereby achieving both high expression level and high biological activity without the complexity and cost of actual eukaryotic systems
3Ease of manufacture
If existing rhBMP-2 sequences are used for recombinant expression, then production process is simple, but purification yield and purity are insufficient
Solution Approach 1:
The patent changes the nucleotide sequence parameters by optimizing codon usage and removing rare codons, which improves the translation efficiency and protein folding in E. coli, thereby enhancing the purification yield and purity while maintaining the simplicity of the prokaryotic production process
Data Source
AI summary
The present invention relates to a field of biotechnology. It discloses a novel amino acid sequence of recombinant human bone morphogenetic protein-2 (rhBMP-2) and encoded nucleotide sequences thereof, and a method for preparing rhBMP-2. In the present invention, by further optimizing the nucleotide sequence of rhBMP-2, novel amino acid sequence of rhBMP-2 that can express good ectopic induced osteogenic activity and has good renaturation and purification effect is screened out. The engineered bacteria constructed by the present invention can induce the production of recombinant rhBMP-2 protein with an expression level of about 55%, and the produced target protein is more easily denatured and purified than the existing recombinant rhBMP-2, with better denaturation and purification effect and higher osteoinductive activity.