Recombinant Protein Production via Oxidizing Cytoplasm

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Solution Overview

Problem

Current methods for producing recombinant proteins at commercial scales often result in insoluble inclusion bodies, requiring additional refolding steps and the use of reducing agents to recover properly folded proteins, which is time-consuming and inefficient, especially for proteins with disulfide bonds.

Innovation Solution

A method involving the expression and purification of proteins as solubilizable complexes in bacterial cells, where proteins are sedimented into a pellet during centrifugation and solubilized without the need for reducing agents, using chaotropic agents like urea and guanidine hydrochloride, allowing for the recovery of properly folded and active proteins.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If recombinant proteins are produced in bacterial cytoplasm, then production quantity and speed are improved, but proteins form insoluble inclusion bodies requiring additional refolding steps

Engineering Contradiction:

Improveproduction quantity and speedVSAvoidprocess complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent modifies the redox potential parameter of the bacterial cytoplasm by introducing disulfide bond catalysts and oxidizing agents, transforming it from a reducing environment to an oxidizing environment. This parameter change enables proper disulfide bond formation during translation, preventing inclusion body formation while maintaining high productivity benefits of cytoplasmic expression.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent introduces intermediary molecules including disulfide bond catalysts (DsbA, DsbC, PDI), oxidizing agents (thioredoxin, glutathione), and chaperone proteins that mediate proper protein folding and disulfide bond formation. These intermediaries facilitate correct protein structure formation during translation, eliminating the need for subsequent refolding steps.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If refolding steps with reducing agents are used to recover soluble protein from inclusion bodies, then properly folded protein is obtained, but time consumption increases

Engineering Contradiction:

Improveprotein folding correctnessVSAvoidtime consumption

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent performs preliminary action by establishing the oxidizing environment and introducing disulfide bond catalysts before protein translation begins. This ensures that disulfide bonds are formed correctly during the translation process itself, preventing inclusion body formation upfront and eliminating the need for time-consuming refolding steps afterward.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent skips the traditional multi-step refolding process by directly producing correctly folded proteins with proper disulfide bonds during translation in the oxidized cytoplasm. This rushing through of the folding process during synthesis eliminates intermediate purification and refolding steps, significantly reducing time consumption.

Inventive Principle:

Principle #21Skipping (Rushing through)

3Stability of the object's composition

If reducing agents are used to convert disulfide bonds to thiol groups, then insoluble aggregates are solved, but additional chemical reagents and steps are required

Engineering Contradiction:

ImprovesolubilityVSAvoidmanufacturing simplicity

Core Design Contradiction:

Stability of the object's compositionVSEase of manufacture

Solution Approach 1:

The patent inverts the traditional approach by instead of using reducing agents to break disulfide bonds after aggregation, it uses oxidizing agents to promote disulfide bond formation during translation. This inversion prevents aggregation in the first place by ensuring correct disulfide bonding, maintaining solubility without requiring reducing agents or additional chemical treatment steps.

Inventive Principle:

Principle #13The other way round (Inversion)

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 method enables the efficient production of properly folded and active recombinant proteins with high yields, eliminating the need for refolding steps and reducing agent use, and achieving recovery rates of up to 80% of the total protein material.

Implementation Method 1

solubilized without the need for reducing agents, using chaotropic agents like urea and guanidine hydrochloride

Methodology Applied

Scientific EffectChaotropic effect:

Implementation Method 2

at least some of said at least one gene product in the first solution can be sedimented by centrifugation (in salt conditions of 200 mM NaCl at pH 7.4 and 4 degrees C.) at a force of: 900×g, or at between 900×g and 7,000×g, or at 7,000×g, to form a solubilizable pellet

Methodology Applied

Scientific EffectCentrifugal separation: Centrifugal Separation

Data Source

PatentUS11584785B2C-peptides and proinsulin polypeptides comprising the same

Publication Date: 2023.02.21 ABSCI CORPORATION

AI summary

A connecting polypeptide has SEQ ID NO:73. A proinsulin polypeptide includes a mature insulin A-chain, a mature insulin B-chain, and a connecting peptide comprising SEQ ID NO: 73 linking the mature A-chain and the mature B-chain, wherein the connecting peptide is not a native human proinsulin C-peptide. The proinsulin polypeptides according to the invention can be made in high titers and in high purity.