Soluble Telomerase Complex Synthesis via Concomitant Cell-Free Assembly
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for synthesizing active full-length human telomerase are inefficient, often resulting in insoluble and low-yield products due to incorrect folding and missing stabilizing components, requiring time-consuming separate synthetic steps and low protein yields.
Innovation Solution
Concomitant in vitro synthesis of the telomerase protein subunit and RNA subunit in a eukaryotic cell lysate reaction medium, allowing self-assembly of the telomerase complex without purification steps, using a dialysis system for continuous supply of reactants and removal of by-products, and optionally including a caspase inhibitor to extend reaction time and increase yields.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If in vivo experiments are used to synthesize human telomerase, then the method is biologically relevant, but the yield of active full-length telomerase is low and the protein is often insoluble
Solution Approach 1:
The patent uses a cell-free translation system as an intermediary between in vivo biological processes and in vitro synthesis. This system incorporates eukaryotic components (ribosomes, tRNAs, factors) that maintain biological relevance while allowing controlled synthesis of soluble telomerase protein without the limitations of living cells
Solution Approach 2:
The patent changes the physical state and environment parameters by moving from intact living cells to a cell-free system with controlled conditions. This includes adjusting ionic strength, adding chaperones, and optimizing translation conditions to improve protein solubility and yield while maintaining biological functionality
2Manufacturing precision
If separate synthesis steps are used for telomerase components, then each component can be optimized individually, but the process is time-consuming and complex
Solution Approach 1:
The patent merges the synthesis of telomerase components (TERT protein and TR RNA) into a single cell-free translation reaction. Both components are synthesized simultaneously in the same reaction vessel, eliminating the need for separate synthesis, purification, and assembly steps while maintaining the ability to optimize each component's expression
3Manufacturing precision
If multiple separate synthetic steps are required, then component purity can be controlled, but the device complexity and number of operations increase
Solution Approach 1:
The patent combines multiple synthetic operations into a single cell-free translation reaction. The system simultaneously synthesizes TERT protein, TR RNA, and facilitates their assembly into functional telomerase complex in one pot, reducing device complexity from multiple separate reaction vessels and purification steps to a single integrated system
4Ease of manufacture
If conventional cell-free systems are used, then the synthesis can be performed in vitro, but the protein yields are not satisfactory
Solution Approach 1:
The patent optimizes in vitro synthesis parameters by adjusting ionic strength, adding specific chaperones (Hsp90, Hsp70), and controlling translation conditions. These parameter changes dramatically improve protein yield and solubility while maintaining the advantages of in vitro synthesis
Solution Approach 2:
The patent introduces molecular chaperones as intermediary proteins that facilitate proper folding and assembly of telomerase components during translation. These chaperones act as mediators between the translation machinery and the telomerase complex, ensuring high-yield production of soluble, functional 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 simplifies the synthesis process, significantly increasing the yield of soluble and active telomerase complex, achieving higher protein production efficiency compared to previous methods.
Implementation Method 1
reactants diffuse through the dialysis membrane out of at least one further compartment, the supply and discharge compartment, into the reaction compartment and reaction by-products diffuse through the dialysis membrane out of the reaction compartment into the supply and discharge compartment
Implementation Method 2
performing a translation reaction to synthesize the telomerase reverse transcriptase subunit (TERT)
Implementation Method 3
self-assembling of the TERT protein subunit and the TR RNA subunit in the reaction medium to form a functional telomerase complex
Data Source
Figure 1~2
AI summary
The present invention relates to a method for preparing a soluble and functional telomerase complex in eukaryotic cell-free systems which comprises at least the following steps: a) providing a reaction medium comprising at least the following components: - a nucleic acid template coding for the telomerase reverse transcriptase subunit (TERT) - a DNA template coding for the intrinsic telomerase RNA (TR), - an RNA polymerase - an eukaryotic cell lysate; b) concomitantly performing a translation reaction to synthesize the telomerase reverse transcriptase subunit (TERT) and a transcription reaction to synthesize the intrinsic telomerase RNA (TR); c) self-assembling of the TERT protein subunit and the TR RNA subunit in the reaction medium to form a functional telomerase complex; d) optionally further purifying and/or isolating the telomerase complex. In more specific embodiments, the telomerase is human telomerase and the eukaryotic cell lysate is selected from the group comprising wheat germ lysates, insect cell lysates, cell extracts from CHO and HEK cells.