Human Elp123 Complex Purification Yield via Microfluidization and Benzonase
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for obtaining and purifying the human Elp123 complex result in low yields, making them unsuitable for downstream applications.
Innovation Solution
An optimized protein purification method involving microfluidization for lysis, addition of benzonase, optional heparin column steps, and size exclusion chromatography using a Superose 6 column is employed to achieve higher yields of functional human Elp123 complex.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard purification method is used, then purification is achieved, but yield is low
Solution Approach 1:
The patent applies preliminary action by adding benzonase enzyme before the main purification steps to pre-digest nucleic acids. This preliminary digestion of genomic DNA and RNA prevents subsequent clogging and loss of protein during chromatography steps, thereby increasing the final yield of purified Elp123 complex without compromising purification quality
Solution Approach 2:
The patent replaces conventional sonication (mechanical lysis) with microfluidization for cell lysis. This substitution provides more efficient and homogeneous cell disruption, leading to better protein recovery and higher yield while maintaining protein integrity and activity throughout the purification process
2Quantity of substance
If conventional lysis method is used, then cell disruption is achieved, but protein recovery is low
Solution Approach 1:
The patent replaces conventional sonication with microfluidization technology for cell lysis. The microfluidizer forces cells through a narrow gap at high pressure, creating efficient mechanical disruption that recovers more protein while maintaining better protein integrity compared to sonication, thus improving both protein recovery and ease of manufacture
3Reliability
If DNase alone is used, then nucleic acid degradation is achieved, but purification quality is insufficient
Solution Approach 1:
The patent introduces benzonase enzyme as an intermediary agent that specifically degrades nucleic acids before purification. This enzymatic digestion acts as a mediator between the crude lysate and the purification columns, removing nucleic acid contamination that would otherwise interfere with downstream applications while preserving the target protein complex
Solution Approach 2:
The patent performs preliminary nucleic acid digestion by adding benzonase to the crude lysate before chromatography steps. This preliminary action degrades genomic DNA and RNA into smaller fragments that do not interfere with subsequent purification steps, ensuring both high purification quality and adequate protein yield
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
The optimized method achieves a yield of 200-300 µg of active Elp123 protein from 3 L cell culture, significantly higher than traditional methods, allowing for effective characterization and potential therapeutic applications.
Implementation Method 1
the lysis procedure using a microfluidizer instead of conventional sonication
Implementation Method 2
the addition of benzonase instead of adding DNase alone
Implementation Method 3
optional step of heparin column to remove excess of nucleic acid contamination
Implementation Method 4
size exclusion purification using a s6 Superose column within the same day
Data Source
AI summary
In subject application a highly efficient method for obtaining and purification of functional human Elp123 complex has been disclosed.