Modified PDI Fusion Partner for Soluble Recombinant Protein Expression

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Solution Overview

Problem

Current methods for producing recombinant proteins in E-coli systems face challenges such as low solubility and folding efficiency, leading to inactive protein forms due to inclusion body formation and inadequate disulfide bond formation, which complicates the refolding process and reduces expression rates.

Innovation Solution

A method involving a genetically modified Protein Disulfide Isomerase (PDI) as a fusion partner, where the ribosome binding site is removed and the M6 sequence (KIEEGK) is added, enhancing expression rates and solubility, and allowing for enzymatic cleavage and purification of the target protein.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If recombinant proteins are produced in E-coli cytoplasm, then high expression rates are achieved, but proteins form inclusion bodies and lose solubility and activity

Engineering Contradiction:

Improveexpression rateVSAvoidprotein solubility and activity

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent uses a fusion partner protein as an intermediary to bridge the target protein and the E-coli expression system. The fusion partner is specifically designed to prevent inclusion body formation while maintaining high expression rates, thereby mediating between productivity and protein quality requirements

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies the fusion partner protein by removing the ribosome binding site and adding the M6 sequence (KIEEGK) at the amino terminus. These parameter changes in the protein sequence optimize both expression efficiency and solubility, resolving the contradiction between high expression and proper folding

Inventive Principle:

Principle #35Parameter changes

2Reliability

If fusion partners are used to improve solubility, then protein folding is enhanced, but expression rates decrease

Engineering Contradiction:

Improveprotein foldingVSAvoidexpression rate

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent optimizes the fusion partner protein by removing the ribosome binding site to prevent premature translation and by adding the M6 sequence (KIEEGK) at the amino terminus. These parameter changes enhance both solubility and expression rate simultaneously, overcoming the traditional trade-off

Inventive Principle:

Principle #35Parameter changes

3Reliability

If refolding procedures are applied to inclusion bodies, then active proteins can be obtained, but time and expense increase significantly

Engineering Contradiction:

Improveactive protein formVSAvoidrefolding time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent performs preliminary action by using the fusion partner protein to prevent inclusion body formation in the first place. By maintaining proper folding during expression, the need for subsequent refolding procedures is eliminated, saving significant time and resources

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent extracts the problematic ribosome binding site from the fusion partner protein sequence. This removal prevents the formation of inclusion bodies by altering translation dynamics, thereby eliminating the need for refolding procedures

Inventive Principle:

Principle #2Taking out (Extraction)

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

This approach stabilizes the production of recombinant proteins in a soluble and active form with high expression rates, improving protein folding and disulfide bonding, and facilitates easy purification, overcoming the limitations of traditional methods.

Implementation Method 1

another reason is why disulfide bonds in the protein should be suitably formed so that the proteins can be biologically active

Methodology Applied

Scientific EffectDisulfide bond formation: Chemical Bonding

Implementation Method 2

the proteins are not exactly folded into the active forms when the eukaryotic proteins are produced in cytoplasm of the prokaryotic E-coli

Methodology Applied

Scientific EffectProtein folding:

Implementation Method 3

adding a sequence lysine-isoleucine-glutamic acid-glutamic acid-glycine-lysine (KIEEGK (SEQ ID NO: 37), referred to as 'M6') to an amino terminus of the normal PDI

Methodology Applied

Scientific EffectTranslation enhancement:

Implementation Method 4

employing a modified PDI as a fusion partner, wherein the modified PDI is obtained by removing a ribosome binding site from a normal PDI by means of a genetic modification and adding a sequence

Methodology Applied

Scientific EffectProteolytic cleavage: Decomposition (biological)

Data Source

PatentUS8080387B2Method for preparing soluble and active recombinant proteins usins PDI as a fusion partner

Publication Date: 2011.12.20 PNP BIOPHARM CO LTD

AI summary

The present invention relates to a method for producing a recombinant protein capable of increasing expression rate of a target protein and also improving solubility and folding of the expressed target protein using a modified protein disulfide isomerase (PDI) as a fusion partner, and an expression vector containing the modified PDI gene as a fusion partner. The method for preparing a recombinant protein using a modified PDI as a fusion partner according to the present invention may solve the problems concerning a low yield and solubility and folding that conventional fusion partners have, and be widely used for protein drug and industrial protein production.