Fibronectin Secretion Leader for Recombinant Polypeptide Yield
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Solution Overview
Problem
Current recombinant polypeptide expression systems often fail to efficiently export polypeptides from host cells, limiting the secretion of recombinant proteins, which is crucial for their production and application, especially in therapeutic contexts.
Innovation Solution
The use of fibronectin secretion leaders, such as mammalian and reptilian fibronectin sequences, operably linked to expression vectors in host cells to facilitate the secretion of recombinant polypeptides, including monoclonal antibodies, by attaching them to the N-terminus of the polypeptide and utilizing appropriate promoters and selectable markers for expression in various host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native signal peptides (PhoA, MalB, OmpA) are used for secretion, then polypeptides can be secreted to the periplasm, but secretion efficiency is insufficient for high-yield production
Solution Approach 1:
The patent changes the key parameter of the secretion leader sequence by replacing native E. coli signal peptides (PhoA, MalB, OmpA) with fibronectin secretion leaders derived from mammalian or reptilian sources. This parameter change fundamentally alters the secretion mechanism and efficiency, enabling high-yield production of recombinant polypeptides in the periplasmic space.
Solution Approach 2:
The fibronectin secretion leader acts as an intermediary element that mediates between the recombinant polypeptide and the secretion machinery. This foreign-derived leader sequence serves as a bridge that enables efficient recognition and transport of the heterologous protein to the periplasm, overcoming the limitations of native signal peptides.
2Productivity
If fibronectin secretion leaders are used, then secretion efficiency and yield are enhanced, but the complexity of the expression system increases due to need for operable linkage and N-terminal attachment
Solution Approach 1:
The fibronectin secretion leader is designed and positioned in advance within the expression cassette, with predetermined operable linkage to the target polypeptide coding sequence. The N-terminal attachment is pre-established through proper reading frame alignment and contiguous nucleotide sequence arrangement, eliminating the need for complex post-expression modifications.
Solution Approach 2:
The secretion leader sequence is merged with the target polypeptide coding sequence in a single operably linked construct. The fibronectin leader and target gene are combined into one continuous nucleotide sequence that ensures proper reading frame and N-terminal attachment, simplifying the overall expression system design.
Data Source
AI summary
A process for the production of a target polypeptide is provided. The process comprises expression of an expression vector for expressing a target polypeptide in a host cell, preferably a mammalian cell, the expression vector comprising an expression cassette comprising a polynucleotide encoding a recombinant polypeptide operably linked to a fibronectin secretion leader sequence; and recovering the target polypeptide.