Recombinant Phosphatase Expression for Disulfide Bond Folding in B. Subtilis
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Solution Overview
Problem
Bacillus subtilis host cells face challenges in efficiently expressing and secreting polypeptides containing disulfide bonds, leading to secretion stress and reduced overall expression and secretion of functional proteins.
Innovation Solution
Developing recombinant Gram-positive host cells, such as B. subtilis, with deletions of endogenous protease genes and expressing recombinant phosphatases containing multiple disulfide bonds, using extrachromosomal vectors or integrating nucleic acids into the host genome to enhance correct folding and secretion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If B. subtilis host cells are used to express polypeptides containing disulfide bonds, then protein secretion capacity is high, but secretion stress response is induced leading to reduced expression and secretion
Solution Approach 1:
The patent extracts and removes endogenous protease genes (aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA, nprB) from the B. subtilis genome to eliminate harmful factors that degrade secreted proteins. This gene deletion strategy removes the secretion stress response mechanism while preserving the high secretion capacity of the host cell.
Solution Approach 2:
The patent introduces heterologous thiol-disulfide oxidoreductase genes (dsbA, dsbC, dsbD) as intermediary enzymes to facilitate proper disulfide bond formation in the secreted polypeptides. These intermediary proteins mediate the oxidation process required for correct folding of disulfide-containing proteins without triggering secretion stress.
2Manufacturing precision
If polypeptides containing multiple disulfide bonds are expressed in B. subtilis, then functional protein production is achieved, but correct folding is compromised due to inefficient disulfide bond formation
Solution Approach 1:
The patent introduces heterologous thiol-disulfide oxidoreductase genes (dsbA, dsbC, dsbD) as intermediary enzymes to facilitate proper disulfide bond formation in the secreted polypeptides. These intermediary proteins mediate the oxidation process required for correct folding of disulfide-containing proteins.
Solution Approach 2:
The patent changes the biochemical environment by introducing exogenous oxidoreductases that alter the redox conditions in the secretory pathway. This parameter change enables efficient disulfide bond formation by providing the necessary oxidative environment that is otherwise insufficient in B. subtilis.
3Stability of the object's composition
If endogenous protease genes are deleted to reduce protein degradation, then protein stability is improved, but host cell complexity increases
Solution Approach 1:
The patent extracts and removes endogenous protease genes (aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA, nprB) from the B. subtilis genome to eliminate harmful factors that degrade secreted proteins. This targeted gene deletion improves protein stability while the modular nature of the genetic modifications keeps the overall system manageable.
Data Source
AI summary
Provided herein, inter alia, are compositions and methods for expressing recombinant phosphatases containing numerous disulfide bonds in host cells.