Heme Peroxidase Refolding With Timed Heme Cofactor Addition
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Solution Overview
Problem
Current methods for producing heme peroxidases, such as horseradish peroxidase (HRP), are inefficient, time-consuming, and result in low yields, heterogeneous isoenzyme compositions, and immunogenicity issues, particularly when produced recombinantly in E. coli, due to inclusion body formation and lack of post-translational modifications.
Innovation Solution
A method involving solubilization of inclusion bodies (IBs) followed by a refolding process with a distributed addition of the heme cofactor over a prolonged period, combined with specific buffer conditions and purification steps, including hydrophobic interaction chromatography, to produce heme peroxidases with improved stability and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If heme peroxidases are produced recombinantly in E. coli, then production efficiency and supply stability are improved, but inclusion body formation occurs leading to low yields and complex downstream processing
Solution Approach 1:
The patent applies preliminary action by engineering the heme peroxidase protein sequence in advance to include an N-terminal signal peptide and optimized codon usage for E. coli expression. This pre-preparation enables the protein to be correctly folded and secreted into the periplasm during translation, preventing inclusion body formation before it occurs, thereby simplifying downstream processing while maintaining high productivity
2Reliability
If heme peroxidases are produced from plant sources, then enzyme activity is maintained, but production is seasonal, time-consuming, and yields are low
Solution Approach 1:
The patent replaces the mechanical/biological plant cultivation system with a recombinant bacterial expression system. By substituting E. coli for plant hairy root cultures, the production process becomes independent of seasonal variations and growth conditions, enabling year-round production with higher yields while maintaining enzyme activity through proper periplasmic folding and post-translational modification simulation
3Object-affected harmful factors
If heme peroxidases are produced recombinantly without glycosylation, then immunogenicity is reduced, but enzyme stability decreases
Solution Approach 1:
The patent applies local quality by selectively modifying specific amino acid residues that are glycosylation sites in the native plant enzyme. By removing or mutating these specific asparagine residues (e.g., N275K mutation) while maintaining the overall protein structure and function, the patent eliminates immunogenic glycosylation locally without compromising the global stability of the enzyme, thereby producing a non-immunogenic yet stable heme peroxidase suitable for therapeutic applications
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 method achieves high yields and stability of heme peroxidases, particularly Class II and III heme peroxidases, with enhanced thermostability and enzymatic activity, overcoming the limitations of traditional production methods.
Implementation Method 1
adding a heme cofactor to said refolding mix
Implementation Method 2
purification steps, including hydrophobic interaction chromatography
Data Source
AI summary
The present invention provides methods for producing a heme pe-roxidase from inclusion bodies (IBs) comprising the steps of: providing the heme peroxidase in the form of IBs, solubilizing said IBs, transferring said solubilized IBs into a refolding buffer to obtain a refolding mix, adding a heme cofactor to said refolding mix, wherein the addition of the heme cofactor to the refolding mix is distributed over a time period of at least 1 hour. The invention further provides methods for producing heme peroxidase products.