Scalable Fermentation for Soluble Bacteriophage Capsid Protein
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Solution Overview
Problem
Existing methods for expressing bacteriophage capsid proteins in bacterial hosts face challenges in scaling up beyond laboratory scale due to reduced yield, formation of insoluble aggregates, and promoter leakage, which affects the quality and purity of virus-like particles (VLPs).
Innovation Solution
A fermentation process using an inducible lactose promoter, overexpression of lacI, and controlled feeding of a major carbon source minimizes promoter leakage and insoluble protein formation, allowing scalable production of recombinant capsid proteins that self-assemble into VLPs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the fermentation process is scaled up from laboratory scale to fermenter volumes of 50 litres and more, then the production capacity increases, but the capsid protein yield diminishes due to increased promoter leakage and lowered plasmid retention
Solution Approach 1:
The patent modifies the promoter system by using a tac promoter instead of traditional inducible promoters, and changes the induction method from IPTG to auto-induction through lactose metabolism. This parameter change in the expression system maintains high protein yield while enabling scalable fermentation up to 50 litres and beyond, resolving the contradiction between production capacity and protein yield.
2Productivity
If high expression rates of recombinant capsid protein are achieved, then the productivity increases, but the formation of incorrectly folded protein species and inclusion bodies increases, hampering downstream processes
Solution Approach 1:
The patent employs a dynamic expression system where the tac promoter provides constitutive expression during growth phase, and induction occurs naturally when cells are exposed to lactose. This dynamic control allows the system to adapt expression rates to cellular conditions, maintaining high productivity while preventing the formation of inclusion bodies and incorrectly folded proteins that occur with constant high-level induction.
Solution Approach 2:
The bacterial host is pre-engineered to contain the tac promoter and necessary regulatory elements before induction. The cells are grown in the presence of lactose from the beginning, allowing gradual induction of protein expression as cells divide and metabolize lactose, rather than sudden high-level induction that causes aggregation and misfolding.
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
The process achieves high yields of soluble capsid proteins with improved plasmid retention and reduced aggregate formation, ensuring consistent quality and purity of VLPs at commercial scales.
Implementation Method 1
cultivating is performed in batch culture and under conditions under which said promoter is repressed by lacI
Implementation Method 2
a promoter being inducible by lactose
Implementation Method 3
capsid proteins of bacteriophages are particularly suited as antigen carrier. They have been shown to efficiently self-assemble into VLPs upon expression in a bacterial host
Data Source
AI summary
This invention provides a robust fermentation process for the expression of a capsid protein of a bacteriophage which is forming a VLP by self-assembly, wherein the process is scalable to a commercial production scale and wherein the expression rate of the capsid protein is controlled to obtain improved yield of soluble capsid protein. This is achieved by combining the advantages of fed-batch culture and of lactose induced expression systems with specific process parameters providing improved repression of the promoter during the growth phase and high plasmid retention throughout the process.