Lysostaphin Secretory Expression in E. coli
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Solution Overview
Problem
Current methods fail to achieve effective secretory expression of lysostaphin in Escherichia coli, which is crucial for developing human drugs due to limitations in existing expression systems and high costs associated with intracellular expression in other hosts like Lactobacillus.
Innovation Solution
Incorporating a signal peptide sequence before the coding sequence of mature lysostaphin to facilitate secretory expression in Escherichia coli, using expression vectors like pUC, pET, or pGEX, and inducing expression with IPTG, resulting in active lysostaphin production in the culture supernatant with reduced host contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If intracellular expression is used in Escherichia coli or other hosts, then lysostaphin can be produced, but the expression level is low and purification is complex
Solution Approach 1:
The patent introduces a signal peptide sequence as an intermediary element between the promoter and the lysostaphin coding sequence. This signal peptide acts as a mediator that directs the expressed protein to the secretory pathway, enabling lysostaphin to be secreted into the culture supernatant rather than remaining intracellular, thereby simplifying purification and increasing production levels
Solution Approach 2:
The patent modifies the expression system by changing the localization parameter of the expressed protein from intracellular to extracellular through the addition of a signal peptide. This parameter change transforms the expression outcome from low-level intracellular accumulation to high-level secretory expression, resolving the contradiction between productivity and ease of manufacture
2Ease of manufacture
If secretory expression is achieved, then purification is simplified and host contamination is reduced, but existing methods fail to achieve effective secretory expression in Escherichia coli
Solution Approach 1:
The signal peptide serves as a reliable intermediary that ensures effective secretory expression in Escherichia coli. By selecting and optimizing appropriate signal peptide sequences, the patent establishes a reliable mechanism for protein secretion, making secretory expression effectively achievable in this host system
Solution Approach 2:
The patent employs feedback mechanisms through the selection and optimization of signal peptide sequences to ensure reliable secretory expression. By testing and selecting signal peptides that effectively direct secretion in Escherichia coli, the system achieves both ease of manufacture and reliability
3Productivity
If other hosts like Lactobacillus are used for expression, then lysostaphin can be produced, but costs are high and the process is complex
Solution Approach 1:
The patent makes the Escherichia coli expression system universal and multi-functional by enabling it to perform secretory expression of lysostaphin. This transforms E. coli from a host suitable only for intracellular expression to one that can also perform efficient secretory expression, matching or exceeding the capabilities of more complex hosts like Lactobacillus while maintaining cost-effectiveness
Solution Approach 2:
The patent utilizes Escherichia coli, a cost-effective and easily cultured host organism, to replace more expensive and complex expression systems. By achieving effective secretory expression in this inexpensive host, the patent eliminates the need to use costly alternatives while maintaining high productivity
Data Source
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AI summary
A method of secretory expression of lysostaphin in Escherichia coli at high level, which comprises constructing a expression vector by cloning a sequence encoding a signal peptide which is suitable for secretory expression in Escherichia coli before part or whole gene sequence which encodes mature lysostaphin, and ligating the cloned sequence with a promoter; and transforming Escherichia coli with the expression vector, culturing and fermenting, and then isolating lysostaphin from the supernatant of the fermentation broth. The advantage of secretory expression is that the expression product can exist in the medium in an active form, and thus does not need the process for renaturation of the inclusion body; it is more easily to purify from the supernatant of the fermentation broth with high rate of recovery; and there is less contamination from the host's proteins.