Yeast Ferritin Fusion Protein Expression
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Solution Overview
Problem
Yeast species such as Saccharomyces, Schizosaccharomyces, Kluyveromyces, Hansenula, Arxula, Schwanniomyces, Candida, Pichia, and Yarrowia often fail to express target proteins at sufficient yield or quality due to low expression rates and undesirable post-translational modifications, including degradation and glycosylation.
Innovation Solution
Coupling the target protein DNA sequence with a ferritin protein DNA sequence to form a composite protein, which is expressed and maintained in the yeast cells, allowing for high expression rates and avoidance of the secretory pathway to prevent undesirable modifications, using host cells like Hansenula polymorpha or Pichia pastoris.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If target proteins are expressed using conventional yeast expression systems, then expression rates are achieved, but undesirable post-translational modifications occur including degradation and glycosylation
Solution Approach 1:
The invention introduces a ferritin fusion partner as an intermediary component that mediates between the target protein and the yeast cellular environment. The ferritin fusion partner acts as a protective intermediary that prevents direct interaction between the target protein and harmful cellular components, thereby preventing degradation and unwanted post-translational modifications while maintaining high expression rates
Solution Approach 2:
The invention creates a composite protein structure by fusing the target protein with a ferritin protein. This composite structure combines the benefits of high-level ferritin expression in yeast with the target protein, creating a stable fusion protein that avoids degradation and unwanted modifications. The composite ferritin-target protein structure leverages the stability and solubility of ferritin to protect the target protein
2Ease of operation
If secretory expression pathway is used for target proteins, then protein secretion occurs, but undesirable post-translational modifications are introduced
Solution Approach 1:
The invention extracts the target protein from the secretory pathway by directing its expression to remain intracellular through the ferritin fusion strategy. By maintaining the fusion protein within the cytosol rather than allowing secretion, the system avoids the harmful post-translational modifications that occur in the secretory pathway while still achieving high expression levels
3Reliability
If conventional expression systems are used for difficult-to-express target proteins, then some expression is achieved, but yields remain insufficient
Solution Approach 1:
The invention merges the highly expressible ferritin gene with the difficult-to-express target protein gene to create a fusion construct. This merging leverages the strong expression characteristics of ferritin in yeast to drive high-level production of the target protein, thereby achieving both reliability of expression and high productivity that were previously unattainable
Data Source
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AI summary
In a method for producing a target protein, (a) a cell culture containing host cells of a yeast species of the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Hansenula, Arxula, Schwanniomyces, Candida, Pichia or Yarrowia is provided, wherein the host cells contain at least one first DNA sequence encoding a heterologous ferritin protein and at least one second DNA sequence encoding the target protein, wherein the second DNA sequence is coupled to the first DNA sequence in such a way that upon expression of the first and second DNA sequences, the target protein is coupled to the previously synthesized ferritin protein, forming a composite protein.Then (b) the host cells are induced, using the first DNA sequence encoding heterologous ferritin protein and the second DNA sequence encoding the target protein, to express the composite protein containing the ferritin protein and the target protein, with the composite protein being retained within the host cells. The composite protein is (c) detached from the cell culture, and then (d) the target protein is separated from the composite protein.