cSPP Gene Expression System for Isotope-Enriched Protein Production
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The production of high-quality isotope-enriched protein samples for NMR studies is costly and inefficient, particularly for larger proteins, due to the need for perdeuteration, which results in reduced protein yield and heterogeneously labeled proteins.
Innovation Solution
A method using a vector with a cspA cold shock promoter, tetR gene, and tet operator to induce mRNA-specific endoribonuclease and target protein expression separately, allowing for efficient production of perdeuterated, 13C-, 15N-enriched proteins with greater than 95% labeling efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If perdeuteration methods are employed to improve NMR data quality, then signal-to-noise ratio and line width are improved, but production cost increases significantly
Solution Approach 1:
The patent divides the protein expression process into two distinct phases: (1) initial expression in standard medium to produce sufficient protein mass, and (2) subsequent purification and isotopic labeling of the purified protein. This segmentation allows isotopic labeling to be performed on concentrated protein rather than requiring expensive isotopic medium throughout expression, dramatically reducing costs while maintaining high NMR data quality
Solution Approach 2:
The patent performs preliminary protein expression and purification in standard, inexpensive medium before applying the expensive isotopic labeling step. By expressing the protein first and purifying it to high concentration, the subsequent isotopic labeling requires minimal medium volume, reducing the overall cost of isotopic reagents while ensuring high labeling efficiency
2Productivity
If target protein is expressed prior to culture condensation, then protein yield increases, but labeling homogeneity decreases due to accumulation of unlabeled protein
Solution Approach 1:
The patent extracts the protein from the culture medium through purification steps before applying isotopic labels. By purifying the protein to high concentration and removing it from the culture medium, then performing isotopic labeling in a separate step with purified protein, the method ensures homogeneous labeling while maintaining high protein yield
Solution Approach 2:
The patent introduces purified protein as an intermediary between expression and isotopic labeling. The protein is first expressed in standard medium, then purified to high concentration, and finally used as the substrate for isotopic labeling. This intermediary step decouples the expression and labeling processes, allowing optimal conditions for each while ensuring homogeneous labeling
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 approach significantly reduces production costs and increases the percentage of labeled protein, enabling high-quality NMR data collection and structure determination at a fraction of the traditional method's cost, while minimizing unlabeled contamination.
Implementation Method 1
induce mRNA-specific endoribonuclease and target protein expression separately
Implementation Method 2
incorporate deuterium into the protein sample, diluting the 1H—1H relaxation networks and increasing 13C and 15N relaxation times
Data Source
AI summary
The present invention is directed to the improved methods for the temporal induction of proteins using the condensed single protein production (cSPP) system.