Truncated Monomeric BChE Codon Optimization
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Solution Overview
Problem
Current methods for producing recombinant butyrylcholinesterase (BChE) face challenges such as low yield, complex manufacturing processes, short half-life, heterogeneous product composition, and high costs, making it difficult to develop the enzyme as a therapeutic agent for conditions like organophosphate poisoning and cocaine intoxication.
Innovation Solution
The development of a codon-optimized nucleic acid encoding a truncated monomeric form of BChE, which is more stable and easier to produce, with optimized glycosylation and sialylation sites, allowing for higher yields and longer serum half-life, thereby reducing the need for expensive post-synthetic modifications like pegylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant BChE is produced using conventional methods, then the enzyme can be obtained for therapeutic use, but the production yield is low and the manufacturing process is complex
Solution Approach 1:
The patent divides the full-length BChE protein (590 amino acids) into a truncated version (amino acids 1-564), removing the C-terminal domain. This segmentation simplifies the protein structure, making it easier to produce recombinantly with higher yields while maintaining the essential catalytic activity for detoxifying organophosphates and cocaine
Solution Approach 2:
The patent modifies the nucleic acid sequence through codon optimization to match human cell preferences, and engineers specific glycosylation sites (N-X-S/T motifs) into the truncated BChE sequence. These parameter changes in the molecular structure enable efficient expression in mammalian cells and improve protein stability and half-life in serum
2Manufacturing precision
If conventional recombinant BChE production methods are used, then the enzyme can be produced, but the product composition is heterogeneous and purification is difficult
Solution Approach 1:
The patent extracts or removes the C-terminal domain (amino acids 565-590) from the full-length BChE protein. This removal eliminates regions that contribute to heterogeneity and complex post-translational modifications, resulting in a more homogeneous product that is easier to purify and characterize for therapeutic applications
3Duration of action of stationary object
If conventional BChE production methods are used, then the enzyme can be obtained, but the serum half-life is short requiring expensive post-synthetic modifications
Solution Approach 1:
The patent performs preliminary engineering of glycosylation sites (N-X-S/T motifs) directly into the truncated BChE sequence during the gene design stage. This preliminary action ensures that when the protein is expressed in mammalian cells, it receives appropriate glycosylation modifications that extend serum half-life, eliminating the need for expensive post-synthetic pegylation or other stabilization modifications
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The truncated monomeric form of BChE is produced in higher quantities, with improved stability and homogeneity, making it a more viable therapeutic agent with extended serum half-life and reduced production costs.
Implementation Method 1
polynucleotides codon-optimized for expression in mammalian, especially human, cells
Implementation Method 2
Butyrylcholinesterase (BChE)...preferentially uses butyrylcholine and benzoylcholine as in vitro substrates...in addition to preventing lethality, the pretreatment prevented behavioral incapacitation...BChE is the major detoxifying enzyme of cocaine...Cocaine is metabolized by three major routes: hydrolysis by BChE
Implementation Method 3
optimized glycosylation and sialylation sites, allowing for higher yields and longer serum half-life
Implementation Method 4
optimized glycosylation and sialylation sites, allowing for higher yields and longer serum half-life
Data Source
AI summary
Isolated nucleic acids encoding polypeptides that exhibit butyrylcholinesterase (BChE) enzyme activity are disclosed, along with molecular criteria for preparing such nucleic acids, including codon optimization. Methods of preparing modified and/or truncated BChE molecules having selected properties, especially selective formation of monomers, are also described. Vectors and cells containing and/or expressing the nucleic acids are also disclosed.