Recombinant Ferritin Expression for Variable Subunit Ratios

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Solution Overview

Problem

Current methods for synthesizing ferritin molecules with varying ratios of heavy chain (FtH) to light chain (FtL) are inefficient, with low recovery, reproducibility issues, and potential for undesired modifications due to oxidation of subunit residues, making it difficult to produce ferritin with predetermined ratios.

Innovation Solution

A method involving the co-insertion of cDNA sequences into a plasmid, transformation into a microbial host, and co-expression of ferritin molecules by exposing the host to varying concentrations of inducers such as IPTG and tetracycline to achieve specific ratios of FtH to FtL subunits.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If in vitro reconstruction methods are used to synthesize heteropolymer ferritin with different H:L ratios, then ferritin molecules with variable subunit ratios can be produced, but the recovery is very low and reproducibility is poor

Engineering Contradiction:

Improveability to produce ferritin with variable H:L ratiosVSAvoidrecovery and reproducibility

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent replaces the mechanical/chemical in vitro reconstruction method with a biological expression system. Instead of denaturing and re-assembling homopolymers through pH adjustment, the invention uses recombinant DNA technology to co-express H and L subunits in host cells, leveraging biological machinery for precise subunit assembly with high recovery and reproducibility

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent controls the H:L ratio by adjusting expression parameters in the biological system. By varying inducer concentrations or using promoters with different strengths, the system achieves precise control over subunit production ratios, transforming the process from low-recovery chemical reassembly to high-reproducibility biological expression

Inventive Principle:

Principle #35Parameter changes

2Productivity

If in vitro reconstruction methods are used to synthesize heteropolymer ferritin, then ferritin molecules can be produced, but the process is tedious and produces low protein yield

Engineering Contradiction:

Improveprotein yieldVSAvoidprocess complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The host cell system performs the ferritin assembly process automatically. Once the recombinant plasmid is introduced, the cellular machinery self-assembles H and L subunits into ferritin molecules with high efficiency, eliminating the need for manual pH adjustment and extensive purification steps required in in vitro methods

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The patent prepares the genetic blueprint in advance through plasmid construction with appropriate promoters and regulators. This preliminary genetic setup enables the host cells to automatically produce ferritin with controlled H:L ratios without requiring complex in vitro reassembly procedures, thereby simplifying the overall process and increasing yield

Inventive Principle:

Principle #10Preliminary action

3Productivity

If in vitro reconstruction methods are used to synthesize heteropolymer ferritin, then ferritin molecules can be produced, but undesired modifications may be introduced due to oxidation of subunit residues

Engineering Contradiction:

Improveferritin productionVSAvoidoxidation of subunit residues

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent employs an inert intracellular environment within the host cell to prevent oxidation. The cellular cytoplasm provides a controlled milieu with low oxygen tension and antioxidant systems that protect ferritin subunits from oxidative damage during assembly, eliminating the need for protective measures in in vitro procedures

Inventive Principle:

Principle #39Inert atmosphere (Inert environment)

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 allows for the efficient and cost-effective production of ferritin with preselected ratios of FtH to FtL, improving yield and reproducibility while minimizing oxidative modifications, enabling the synthesis of ferritin molecules with precise subunit ratios.

Implementation Method 1

co-expressing a homopolymer ferritin molecule or a heteropolymer ferritin molecule at variable ratios of FtH to FtL

Methodology Applied

Scientific EffectTranscription and translation:

Implementation Method 2

exposing the genetically modified microbial host to a variable concentration of a first inducer and a variable concentration of a second inducer

Methodology Applied

Scientific EffectInducer-operator-promoter interaction:

Data Source

PatentUS20230348916A1Expression and purification of recombinant human isoferritins using a novel expression system

Publication Date: 2023.11.02 THE RES FOUND FOR SUNY

AI summary

The present disclosure relates to methods of making of one or more bioengineered ferritin proteins having a preselected ratio of H-subunits (ferritin heavy chain (FTH or H)) to L-subunits (ferritin light chain (FTL or L)). The disclosure further relates to cDNAs, vectors, and host cells for forming the bioengineered ferritin proteins of the present disclosure.