HRP Polypeptide Mutations for Soluble E. coli Expression
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Solution Overview
Problem
Current methods for producing horseradish peroxidase (HRP) recombinantly in E. coli face challenges such as low yields, instability, and compromised enzymatic activity due to inclusion body formation and lack of post-translational modifications, limiting its industrial applications.
Innovation Solution
Introduce specific amino acid exchanges at positions P146 and/or N275 of the HRP sequence, particularly combining mutations like P146Q and N275K, to enhance thermostability and enzymatic activity, enabling high-yield production in E. coli with improved stability and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If HRP is produced recombinantly in E. coli, then steady supply and defined preparation are achieved, but yields are low and enzyme stability is decreased
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations (P146Q, P146A, P146R, P146V, P146E at position 146; and N275K, N275R, N275D, N275S, N275Q, N275A, N275E at position 275) to the HRP protein sequence. These mutations modify the protein's biochemical parameters to enhance both stability and activity while maintaining compatibility with E. coli production systems.
Solution Approach 2:
The patent creates a composite protein structure by combining the HRP core protein with specific amino acid modifications at critical positions. This composite approach integrates the benefits of recombinant production with stabilized protein structure, achieving both high yield and enhanced stability in the engineered enzyme.
2Object-affected harmful factors
If HRP is produced in E. coli, then glycosylation is avoided (reducing immunogenicity), but protein stability is reduced
Solution Approach 1:
The patent compensates for the lack of glycosylation-induced stability through parameter changes in the protein's amino acid sequence. The mutations at positions 146 and 275 specifically address stability issues that would otherwise result from producing unglycosylated HRP in E. coli, thereby maintaining protein stability while avoiding immunogenic glycosylation.
3Ease of manufacture
If wild-type HRP is produced in E. coli, then recombinant production is achieved, but inclusion body formation occurs due to reducing conditions
Solution Approach 1:
The patent modifies the protein's structural parameters through amino acid mutations to enhance its solubility and reduce aggregation propensity. The mutations at P146 and N275 specifically address the inclusion body formation issue by changing the protein's interaction properties, thereby improving soluble protein yield while maintaining ease of recombinant production in E. coli.
4Reliability
If signal sequence is added for periplasmic translocation, then enzyme activity is maintained, but yields are lower and activity may be compromised by translocation tag
Solution Approach 1:
The patent achieves high yield production by modifying the enzyme's intrinsic properties through amino acid mutations rather than relying on periplasmic translocation. The mutations at positions 146 and 275 enhance the enzyme's stability and activity in the cytoplasmic environment, eliminating the need for signal sequences and avoiding the yield and activity compromises associated with translocation tags.
Data Source
AI summary
The present invention provides a polypeptide having peroxidase activity and comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3, wherein said amino acid sequence comprises at least one amino acid exchange compared to SEQ ID NO: 1, wherein said at least one amino acid exchange is an exchange of the amino acid P146 or of the amino acid N275 of SEQ ID NO: 1. The invention further relates to a nucleic acid molecule comprising a sequence encoding said polypeptide, an expression vector comprising said nucleic acid molecule, and a host cell comprising said expression vector. Moreover, methods for producing said polypeptide and compositions and kits comprising said polypeptides are provided.