Surface-Displayed Fusion Proteins for Enzymatic Modification

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Solution Overview

Problem

There is an unmet need for engineered eukaryotic cells that can efficiently enzymatically modify secreted recombinant proteins and other chemicals in a culturing medium, particularly for surface-displayed enzymes that can perform post-translational modifications or utilize alternate carbon sources without releasing enzymes into the medium.

Innovation Solution

Engineered eukaryotic cells expressing surface-displayed fusion proteins with a catalytic domain of an enzyme and an anchoring domain of a glycosylphosphatidylinositol (GPI)-anchored protein, where the anchoring domain comprises at least 200 amino acids and/or 30% serines or threonines, allowing for effective surface display and enzymatic activity, such as deglycosylation or carbon source conversion.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If enzymes are secreted into the culturing medium to modify proteins, then enzymatic modification capability is improved, but enzyme removal complexity and production cost increase

Engineering Contradiction:

Improveenzymatic modification capabilityVSAvoidenzyme removal complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The cell surface itself serves as the platform for enzyme display, eliminating the need for separate enzyme addition and removal steps. The anchor domain mediates self-anchoring of the fusion protein to the cell membrane via GPI attachment, enabling the system to serve its own display function without external assistance.

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The enzyme is extracted from its traditional soluble secreted form and repositioned to the cell surface through fusion with the anchor domain. This extraction of the enzyme from the medium and placement on the cell surface resolves the contradiction by maintaining catalytic function while eliminating the need for enzyme removal.

Inventive Principle:

Principle #2Taking out (Extraction)

2Ease of manufacture

If a short anchor domain is used in the fusion protein, then protein expression simplicity is improved, but surface display efficiency and enzymatic activity decrease

Engineering Contradiction:

Improveprotein expression simplicityVSAvoidsurface display efficiency

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The anchor domain parameters were optimized by increasing its length to at least 200 amino acids and ensuring at least 30% of residues are serines or threonines. These parameter changes enhance O-glycosylation capacity and cell surface display efficiency while maintaining the fusion protein expression system.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The fusion protein combines the enzyme catalytic domain with a composite anchor domain that includes multiple functional elements: GPI attachment signal, O-glycosylation sites (serine/threonine rich regions), and cell surface anchoring capability. This composite structure achieves both simple expression and high surface display efficiency.

Inventive Principle:

Principle #40Composite materials

3Productivity

If the anchoring domain is extended to increase surface display, then enzymatic activity is improved, but fusion protein complexity increases

Engineering Contradiction:

Improveenzymatic activityVSAvoidfusion protein complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The fusion protein is segmented into distinct functional domains: the enzyme catalytic domain and the anchor domain. This segmentation allows the anchor domain to be independently optimized for surface display function (length and amino acid composition) without affecting the enzyme's catalytic domain structure or activity.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The anchor domain serves multiple functions simultaneously: it provides cell surface anchoring, mediates GPI attachment, enables O-glycosylation for stability, and facilitates proper protein folding and trafficking. This multi-functionality achieves high enzymatic activity without requiring multiple separate protein components.

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 enables efficient enzymatic modification of secreted proteins and utilization of alternate carbon sources, reducing the need for separate enzyme removal and enhancing protein production efficiency, while maintaining the proteins' functionality and purity.

Implementation Method 1

an anchoring domain of a glycosylphosphatidylinositol (GPI)-anchored protein

Methodology Applied

Scientific EffectGPI anchoring:

Implementation Method 2

the serines or threonines in the anchoring domain are capable of being O-mannosylated

Methodology Applied

Scientific EffectO-mannosylation:

Data Source

PatentUS20240084243A1Surface displayed fusion proteins

Publication Date: 2024.03.14 CLARA FOODS

AI summary

The present disclosure provides engineered eukaryotic cells comprising a surface displayed fusion proteins comprising a catalytic domain of an enzyme and an anchoring domain of a glycosylphosphatidylinositol (GPI)-anchored protein and methods of use.