Reversible Zinc-Binding Intein for Controlled Protein Purification
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Solution Overview
Problem
Current self-cleaving intein systems face challenges in controlling the self-release step, particularly in mammalian cell culture expression systems, due to premature activation and incompatibility with certain protein bonds, leading to suboptimal target protein yields and prolonged process times.
Innovation Solution
Development of controllable intervening protein sequences (CIPS) incorporating a reversible zinc-binding motif and an intein, allowing for selective and reversible inactivation of the intein using zinc concentrations, pH changes, or chelating agents to control splicing activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If self-cleaving intein systems are used for protein purification, then tag removal is facilitated and purification efficiency is improved, but premature activation occurs during expression leading to suboptimal target protein yields
Solution Approach 1:
The invention modifies the intein system by introducing a pH-sensitive element that changes the intein's splicing activity based on environmental pH conditions. During expression at neutral pH, the intein remains inactive preventing premature cleavage. For tag removal, the system transitions to acidic pH conditions that activate intein splicing, enabling efficient purification without compromising target protein yield.
2Ease of operation
If self-cleaving intein systems are used, then the self-release step is simplified, but the process time is prolonged due to uncontrollable activation kinetics
Solution Approach 1:
The invention introduces a dynamic control mechanism where the intein's splicing activity is modulated by pH changes rather than being static or constitutively active. This allows the system to transition between inactive (during expression) and active (during purification) states, enabling precise temporal control over the self-release step and optimizing overall process time.
3Ease of manufacture
If conventional intein systems are used in mammalian cell culture, then protein expression is achieved, but incompatibility with certain protein bonds occurs leading to suboptimal yields
Solution Approach 1:
The invention applies local modification to the intein structure by introducing pH-sensitive elements at specific positions within the intein sequence. This localized modification allows the intein to maintain compatibility with mammalian cell culture expression systems while simultaneously enabling pH-dependent activation for efficient tag removal, thereby improving target protein yields without sacrificing expression compatibility.
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
Enables efficient and controlled protein purification by suppressing cleavage during expression and inducing rapid self-cleavage post-purification, enhancing the applicability of inteins in mammalian cell culture systems and improving protein yield and purity.
Implementation Method 1
the CIPS comprises a reversible zinc-binding motif and an intein
Data Source
AI summary
Disclosed are compositions comprising an engineered intein designed such that the self-cleaving activity of the intein can be modulated by a zinc-binding motif as well as methods and systems for making and using the compositions.