Split Intein Mediated Ultra-Rapid Protein Purification
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Solution Overview
Problem
Current protein purification methods using inteins are inefficient due to slow cleavage rates and the risk of premature in vivo cleavage, which limits their application in large-scale industrial processes and requires costly proteases for tag removal.
Innovation Solution
The development of a split intein-mediated ultra-rapid purification method (SIRP) using a naturally split DnaE intein with a mutation that suppresses N-terminal cleavage and enhances C-terminal cleavage efficiency, allowing for rapid and specific release of proteins from intein fusion complexes without the need for proteases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional intein-mediated purification methods are used, then protein purification can be achieved, but the cleavage rate is slow and premature in vivo cleavage occurs
Solution Approach 1:
The intein is divided into two separate fragments: an N-terminal fragment and a C-terminal fragment. These fragments are expressed as fusion proteins with different proteins (Intein N-fragment fused to Protein A, Intein C-fragment fused to Protein B). The segmentation prevents premature cleavage while enabling controlled reconstitution and rapid cleavage when fragments are brought together through affinity purification.
Solution Approach 2:
The N-terminal and C-terminal intein fragments are pre-expressed as fusion proteins with target proteins before the purification process. This preliminary expression ensures that the intein fragments are already in place and ready for rapid cleavage upon reconstitution, eliminating the need for protease treatment and reducing overall purification time.
2Ease of manufacture
If proteases are used for tag removal, then complete tag removal can be achieved, but the process becomes costly and time-consuming
Solution Approach 1:
The intein fragments perform self-cleavage when reconstituted, removing the need for external proteases. The intein's intrinsic autocatalytic activity enables it to cleave itself from the target protein after the N- and C-terminal fragments associate,实现ing tag removal through self-service without additional enzymatic steps.
Solution Approach 2:
The mechanical/enzymatic system of protease-mediated tag removal is replaced with the chemical/self-catalytic system of intein autocleavage. This substitution eliminates the need for expensive proteases and simplifies the purification workflow by utilizing the intein's inherent catalytic capability.
3Productivity
If affinity tags are used for protein purification, then purification can be achieved, but the tags remain attached to the purified protein
Solution Approach 1:
The affinity tag (intein) is extracted from the final purified protein product through self-cleavage. After the intein fragments reconstitute and bind to the target protein via affinity interaction, the intein subsequently cleaves itself off, leaving the target protein free of tags. This extraction ensures high protein purity without residual tag contamination.
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 enables the purification of tagless proteins in under 1 hour with high yields and eliminates the risk of premature cleavage, making it suitable for large-scale industrial applications and reducing costs associated with protease use.
Implementation Method 1
contacting a first fusion protein comprising the POI fused to the C-terminus of an intein C-fragment with a second fusion protein comprising an intein N-fragment and a purification tag to form a complex between the first fusion protein and the second fusion protein
Implementation Method 2
cleaving the POI from the intein C-fragment, wherein the protein is released from the complex
Data Source
AI summary
The present invention includes methods, compositions, uses, and kits for purifying a protein of interest (POI) comprising contacting a first fusion protein comprising the POI fused to the C-terminus of an intein C-fragment with a second fusion protein comprising an intein N-fragment and a purification tag to form a complex between the first fusion protein and the second fusion protein, cleaving the POI from the intein C-fragment, wherein the protein is released from the complex; and isolating the POI; the present invention also includes fusion proteins and vector.