Chimeric Gene Design for Recombinant Antimicrobial Peptide Production

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Solution Overview

Problem

The challenge lies in obtaining sufficient amounts of antimicrobial peptides from natural sources, as extraction from animal tissues is limited, prompting the need for alternative production methods like chemical synthesis or genetic engineering to produce recombinant peptides effectively.

Innovation Solution

A chimeric gene encoding the recombinant version of Ap-S (rAp-S) is designed and expressed in E. coli and Nicotiana tabacum, enabling large-scale production of biologically active antimicrobial peptides with antifungal and bactericidal activity, overcoming the limitations of natural extraction methods.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If extraction from animal tissues is used, then natural antimicrobial peptides can be obtained, but the yield is limited and insufficient for large-scale applications

Engineering Contradiction:

Improveyield of antimicrobial peptidesVSAvoiddifficulty of extraction from tissue

Core Design Contradiction:

Quantity of substanceVSEase of manufacture

Solution Approach 1:

The patent creates a chimeric gene that copies the amino acid sequence of natural Ap-S peptide and expresses it in heterologous systems (E. coli and plants), producing recombinant versions (rAp-S, rAp-S-6xHis) that replicate the antimicrobial function without requiring tissue extraction

Inventive Principle:

Principle #26Copying

Solution Approach 2:

The patent replaces the mechanical/biological extraction process from animal tissues with a genetic engineering approach, using chimeric genes and expression vectors to produce peptides through biological synthesis in cultured cells, thereby eliminating the need for tissue harvesting and extraction

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Quantity of substance

If chemical synthesis is used, then antimicrobial peptides can be produced, but the cost is high and scalability is limited

Engineering Contradiction:

Improveproduction volume of peptidesVSAvoidcost of chemical synthesis

Core Design Contradiction:

Quantity of substanceVSEase of manufacture

Solution Approach 1:

The patent employs self-assembling peptide sequences with amphipathic properties that automatically form functional structures (micelles, membranes) when expressed, eliminating the need for complex chemical synthesis steps and post-production processing

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The patent modifies the peptide sequence by adding a 6xHis tag to the C-terminus, which changes the physical properties of the peptide to enable easier purification through nickel affinity chromatography, thereby simplifying the manufacturing process and reducing costs

Inventive Principle:

Principle #35Parameter changes

3Productivity

If recombinant expression is implemented, then large-scale production is achieved, but the complexity of gene design and expression system increases

Engineering Contradiction:

Improveproduction scale of peptidesVSAvoidcomplexity of chimeric gene construction

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent divides the peptide sequence into functional segments: an N-terminal region with amphipathic properties for antimicrobial activity, and a C-terminal 6xHis tag for purification, allowing independent optimization of each segment's function

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent uses a chimeric gene as an intermediary that combines the Ap-S peptide coding sequence with a 6xHis tag sequence, enabling the expression system to produce a fusion protein that possesses both the antimicrobial function of Ap-S and the purification advantage of the His tag

Inventive Principle:

Principle #24Intermediary (Mediator)

4Reliability

If natural Ap-S peptide is used, then antimicrobial activity is achieved, but cytotoxicity to fish cells occurs

Engineering Contradiction:

Improveantimicrobial activity of peptideVSAvoidcytotoxic effect on fish cells

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent modifies the local composition of the peptide by adding a 6xHis tag at the C-terminus, which alters the distribution of hydrophobic and hydrophilic residues, thereby reducing cytotoxicity while preserving antimicrobial activity

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent changes the amino acid sequence parameters by introducing the 6xHis tag, which modifies the peptide's amphipathic balance and reduces its interaction with mammalian cell membranes, thereby decreasing cytotoxicity while maintaining efficacy against pathogens

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentUS10597430B2Chimeric gene for heterologous expression which encodes for peptides with antimicrobial activity

Publication Date: 2020.03.24 INST DE INVESTIGACIONES AGROPECUARIAS

AI summary

The present invention relates to a chimeric nucleotide sequence encoding peptides with antimicrobial activity, to be expressed on plants as bioreactors, plant cell or transformed plant material and E. coli, where plants and bacteria were used for scale-up production of antimicrobial peptide and that produces the peptide sequence derived from nucleotide sequence SEQ ID No. 1, SEQ ID No. 6 and SEQ ID No. 12. It includes a method for obtaining the amino acids sequence SEQ ID No. 9 derived from a chimeric nucleotide sequence which encodes to antimicrobial peptides, wherein said amino acids sequence is derived from the synthetic DNA sequence SEQ ID No. 1, obtained from a back-translation of the amino acid sequence of the peptide Ap-S of Argopecten purpuratus scallop.