Membrane-Bound Reporter Molecules for High-Throughput Cell Sorting
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Solution Overview
Problem
Current methods for selecting high producer cells for recombinant protein manufacturing are labor-intensive, time-consuming, and limited in capacity, as they rely on manual cloning and require fluorescent reporters or other cumbersome techniques.
Innovation Solution
The use of nucleic acid molecules encoding biotin mimetic or biotin acceptor peptides anchored to the cell membrane, which are labeled with fluorescently tagged biotin-binding proteins for sorting by flow cytometry, allowing for efficient identification and isolation of high producer cells based on protein secretion levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If manual single cell cloning techniques are used for selection of high producer cells, then cell selection can be performed, but the process becomes tedious, labor intensive and time consuming with limited screening capacity
Solution Approach 1:
The patent replaces manual mechanical cell picking and sorting methods with flow cytometry-based automated cell sorting. By introducing fluorescent reporters (GFP, RFP, DsRed) that can be detected by flow cytometry, the system enables automated identification and sorting of high-producing cells based on fluorescence intensity, thereby eliminating tedious manual operations and significantly increasing screening capacity to thousands of cells per second
Solution Approach 2:
The patent utilizes fluorescent proteins (green fluorescent protein GFP, red fluorescent protein RFP, dsRed) as reporters that emit different colors/fluorescence intensities. High-producing cells exhibit stronger fluorescence signals correlated with their productivity levels, enabling visual and automated differentiation of cell populations based on color intensity rather than manual assessment
2Productivity
If fluorescent reporters are used for cell sorting by flow cytometry, then rapid cell selection is enabled, but additional reporter genes and staining procedures are required
Solution Approach 1:
The patent merges the reporter gene system with the protein secretion pathway by using secreted fluorescent proteins. The reporter protein is co-secreted with the target protein through the same secretory pathway, allowing simultaneous detection of both proteins' expression levels. This integration eliminates the need for separate intracellular reporter systems and simplifies the overall methodology
Solution Approach 2:
The patent employs fluorescent proteins that serve multiple functions: they act as reporters for flow cytometry detection, can be detected in both intracellular and extracellular compartments, and their secretion status provides additional information about protein folding and secretion efficiency. The same fluorescent reporter system can be applied to various cell types and target proteins
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 enables the rapid and efficient selection of high producer cells, increasing protein productivity by up to 13.5-fold through the correlation of BMP or BAP expression with protein secretion levels, while being applicable to various cell types and conforming to animal component-free conditions.
Implementation Method 1
coupling protein of interest (POI) expression to that of a cell surface membrane protein/peptide marker labeled with biotin mimetic or biotin acceptor peptides
Implementation Method 2
labeled with a fluorescent reporter gene product
Data Source
AI summary
The present invention relates to nucleic acid molecules comprising a nucleic acid sequence encoding a membrane-bound biotin mimetic peptide (BMP) or biotin acceptor peptide (BAP). The invention also relates to a method for selection of high producer cells secreting a protein of interest.