Non-Toxic Protease Cysteine 430 Mutation to Limit Aggregation

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Solution Overview

Problem

Existing methods for producing non-toxic proteases using recombinant microorganisms face significant challenges due to irreversible protein aggregation, leading to low recovery rates and reduced productivity during the refolding process.

Innovation Solution

Introduce a point mutation at amino acid position 430 of the non-toxic protease, substituting cysteine with another amino acid, and fuse it with functional peptides to inhibit irreversible aggregation and enhance refolding efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If non-toxic protease is produced using recombinant microorganisms, then productivity is improved, but irreversible protein aggregation occurs leading to low recovery rates

Engineering Contradiction:

ImproveproductivityVSAvoidrecovery rate

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies parameter changes by introducing a point mutation at amino acid position 430 (Cysteine to Serine substitution) in the non-toxic protease sequence. This molecular parameter change prevents irreversible protein aggregation during expression in recombinant microorganisms, thereby improving recovery rates while maintaining productivity. The mutated protease maintains its enzymatic activity while avoiding inclusion body formation.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If point mutation is introduced to prevent aggregation, then recovery rate is improved, but protein structure may be altered

Engineering Contradiction:

Improverecovery rateVSAvoidprotein structure

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The patent applies local quality by making a targeted point mutation only at amino acid position 430 (Cysteine to Serine substitution) rather than altering the entire protein structure. This localized change specifically addresses the aggregation issue at the critical residue while preserving the overall protein structure and enzymatic activity of the non-toxic protease domain.

Inventive Principle:

Principle #3Local quality

3Manufacturing precision

If complex purification process is used to dissolve and purify inclusion bodies, then purity is improved, but protein structure changes leading to loss of activity

Engineering Contradiction:

ImprovepurityVSAvoidprotease activity

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent applies the taking out principle by extracting and removing the problematic Cysteine residue at position 430 that causes irreversible aggregation. This eliminates the need for complex purification processes involving urea, guanidium chloride, or other denaturing agents. The mutated protease is produced in a soluble, active form directly, avoiding structural changes and activity loss associated with complex refolding procedures.

Inventive Principle:

Principle #2Taking out (Extraction)

Data Source

PatentUS12385028B2Non-toxic protease having improved productivity

Publication Date: 2025.08.12 NC BIT INC

AI summary

The present invention relates to a mutated non-toxic protease in which the amino acid cysteine (Cys) at position 430 of a non-toxic protease represented by the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than cysteine. According to the present invention, it is possible to recover a refolded non-toxic protease from an insoluble fraction from which the non-toxic protease was almost impossible to recover in the prior art, and thus it is possible to produce the non-toxic protease with significantly improved productivity.