Hybrid Cell-Free Protein Synthesis Reagents for Microfluidic Devices

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Solution Overview

Problem

Existing cell-free protein synthesis (CFPS) systems face challenges such as variable and unreliable protein expression yields, limited capability for post-translational modifications, and biofouling issues on hydrophobic surfaces in microfluidic devices.

Innovation Solution

The development of optimized cell-free protein synthesis reagents and methods that combine cell lysates with purified recombinant elements, allowing for enhanced protein expression yields and post-translational modifications, while minimizing biofouling on microfluidic devices.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If cell lysate is used for protein synthesis, then protein expression level is improved, but reliability of expression is worsened due to variable outcomes

Engineering Contradiction:

Improveprotein expression levelVSAvoidexpression consistency

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent combines cell lysate with purified recombinant elements to create a hybrid CFPS system. This merging allows the system to benefit from both high protein expression levels (from cell lysate) and consistent, reliable expression (from purified components), thereby resolving the contradiction between productivity and reliability.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent optimizes the composition and ratios of components in the CFPS system by changing parameters such as the proportion of cell lysate to purified elements, addition of specific supplements, and adjustment of reaction conditions. These parameter changes enable the system to achieve both high expression levels and consistent reliability.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If purified component mixtures are used, then reliability of expression is improved, but protein expression level is worsened

Engineering Contradiction:

Improveexpression consistencyVSAvoidprotein expression level

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent merges purified recombinant elements with cell lysate to create a hybrid system. This combination allows the system to maintain the reliability and consistency of purified components while achieving the high protein expression levels typically associated with cell lysate, thus resolving the contradiction between reliability and productivity.

Inventive Principle:

Principle #5Merging (Combining)

3Ease of operation

If cell-free protein synthesis is performed on hydrophobic surfaces, then device functionality is improved, but biofouling increases

Engineering Contradiction:

Improvedevice functionalityVSAvoidbiofouling

Core Design Contradiction:

Ease of operationVSObject-affected harmful factors

Solution Approach 1:

The patent introduces an intermediary substance (such as a surfactant or coating agent) between the hydrophobic device surface and the biological components in the CFPS system. This intermediary layer prevents direct contact and adsorption of proteins and nucleic acids to the hydrophobic surface, thereby reducing biofouling while maintaining device functionality.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent converts the harmful effect of hydrophobic surfaces (which cause biofouling) into a beneficial property by using the hydrophobicity to repel biological molecules, thereby preventing their adsorption and denatureation on the device surface.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

4Adaptability or versatility

If CFPS system is used for post-translational modification, then protein functionality is improved, but system complexity increases

Engineering Contradiction:

Improvepost-translational modification capabilityVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent creates a universal CFPS system that can perform multiple functions including protein synthesis and post-translational modifications (such as glycosylation, phosphorylation, and proteolytic cleavage) using a single integrated reagent composition. This multi-functionality reduces the need for separate systems, thereby managing complexity while enhancing versatility.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 results in more consistent and higher protein expression yields, along with the ability to perform post-translational modifications, thereby improving the utility of CFPS systems, especially on electrowetting-on-dielectric (EWoD) devices.

Implementation Method 1

electrowetting-on-dielectric (EWoD), and electrokinesis in general have only found limited uses in cell-free biological-based applications

Methodology Applied

Scientific EffectElectrowetting: Electrowetting

Data Source

PatentUS20250146041A1Methods for cell free protein synthesis and post translational modification of the expressed proteins

Publication Date: 2025.05.08 NUCLERA LTD

AI summary

Provided herein are methods of cell-free protein synthesis, optimised cell-free protein synthesis (CFPS) reagents and the post translational modification of the expressed proteins, to increase protein expression yields. The methods are applicable to protein expression on a microfluidic device having hydrophobic surfaces by merging droplets on the device in order to screen a selection of expression and post translational modification compositions in parallel.