Modulating Host Cell Protease Expression for Recombinant Protein Yield
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Solution Overview
Problem
Current methods for producing recombinant proteins in bacteria often result in improperly folded, aggregated, or inactive proteins due to inefficiencies in protein processing and degradation, leading to reduced yields and quality.
Innovation Solution
A library of genetically modified host cell populations is created to modulate the expression of target genes involved in protein production and degradation, allowing for rapid identification of optimal strains for producing properly processed heterologous proteins, including the use of Pseudomonas fluorescens and E. coli strains with modified protease expression and protein folding modulators.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant proteins are produced in bacteria using current methods, then production efficiency is improved, but protein quality deteriorates due to improper folding and aggregation
Solution Approach 1:
The patent modifies host cell parameters by introducing genetically engineered strains with altered protease expression levels and enhanced protein folding capabilities. This changes the biochemical environment parameters to favor proper protein folding while maintaining high production efficiency.
Solution Approach 2:
The patent uses secretion signals as intermediary elements to direct heterologous proteins through the Sec-type secretion system. This intermediary mechanism enables proper protein processing and translocation, improving both quality and yield simultaneously.
2Ease of operation
If secretion signals are used to express recombinant polypeptides, then protein translocation is improved, but production complexity increases
Solution Approach 1:
The patent employs universal secretion signal sequences that can be applied to multiple different heterologous proteins across various expression systems. This multi-functional approach simplifies the overall complexity by using a standardized mechanism rather than protein-specific translocation methods.
3Reliability
If host cell proteases are present during heterologous protein expression, then cellular metabolism is maintained, but protein degradation increases reducing final yield
Solution Approach 1:
The patent converts the harmful effect of proteases into a beneficial selection mechanism by using protease-deficient mutant strains. These strains naturally select for heterologous proteins that are resistant to degradation, thereby improving final yield while maintaining cellular metabolic functions through the use of essential proteases in controlled amounts.
Data Source
AI summary
The present invention provides an array for rapidly identifying a host cell population capable of producing a heterologous recombinant protein with improved yield and/or quality. The array comprises one or more host cell populations that have been genetically modified to increase the expression of one or more target genes involved in protein production, decrease the expression of one or more target genes involved in protein degradation, or both. One or more of the strains in the array may express the heterologous recombinant protein of interest in a periplasm compartment or may secrete the heterologous recombinant protein extracellularly through an outer cell wall. The strain arrays are useful for screening for improved expression of any protein of interest including therapeutic proteins, hormones, growth factors, extracellular receptors or ligands, proteases, kinases, blood proteins, chemokines, cytokines, antibodies and the like.