Extracellular Brazzein Secretion for Higher Yield Purification
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Solution Overview
Problem
Existing methods for producing Brazzein, a high-potency thermostable sweet protein, face challenges such as low expression yields and cumbersome downstream processing, particularly when localized in the periplasmic space of E. coli, making commercial production impractical.
Innovation Solution
A process for extracellular secretion of Brazzein from E. coli cells involving ligation of a signal leader sequence-conjugated nucleotide sequence to Brazzein in a recombinant vector, followed by transformation, culture, and induction with IPTG or lactose, allowing for efficient separation and purification of Brazzein from the culture medium.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If Brazzein is expressed in the periplasmic space of E. coli using the pelB leader sequence, then the protein can be localized to a specific compartment, but the expression yield is low and downstream processing becomes cumbersome requiring multiple centrifugation steps and osmotic shock treatment
Solution Approach 1:
Instead of localizing Brazzein to the periplasmic space and then extracting it through complex osmotic shock procedures, the invention inverts the approach by directing Brazzein secretion directly to the extracellular space through modification of the signal leader sequence. This allows the protein to be naturally exported outside the cell, eliminating the need for osmotic shock treatment and multiple centrifugation steps, thereby simplifying downstream processing while maintaining high expression yields
2Quantity of substance
If Brazzein is expressed intracellularly or in the periplasmic space, then the protein can be produced, but isolation and purification require expensive and inconvenient downstream processes with yield loss
Solution Approach 1:
The invention extracts the signal leader sequence responsible for periplasmic targeting and replaces it with a modified sequence that directs Brazzein secretion to the extracellular space. This extraction of the protein from the cellular compartment eliminates the need for complex downstream processing steps such as cell lysis, periplasmic extraction, and multiple purification steps, thereby reducing both the complexity of the process and the loss of protein yield
3Reliability
If natural extraction methods are used from Pentadiplandra brazzeana, then Brazzein can be isolated, but the process is expensive and not commercially feasible due to geographical limitations
Solution Approach 1:
Instead of extracting Brazzein from its natural source Pentadiplandra brazzeana, which is geographically limited and expensive, the invention creates a recombinant copy of the Brazzein gene and expresses it in E. coli bacteria. This microbial production system can be scaled up commercially without geographical constraints, significantly reducing extraction costs while maintaining reliable production of authentic Brazzein protein
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
This method achieves enhanced yield of Brazzein, ranging from 0.5 g/l to 5 g/l, with high purity and stability, simplifying downstream processing and making large-scale production feasible.
Implementation Method 1
a) ligating a signal leader sequence-conjugated nucleotide sequence to Brazzein in a recombinant vector... to obtain secretion of brazzein in the culture medium
Implementation Method 2
inducing protein expression with inducing agents selected from IPTG or lactose
Implementation Method 3
separating culture medium from cells after 4 to 72 hours post induction
Data Source
AI summary
The present invention discloses a process for the secretion of brazzein in improved yield.