Bacterial Microcompartment Shell Assembly via Proteolytic Cleavage
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Solution Overview
Problem
Current methods for in vitro assembly of bacterial microcompartments (BMCs) face challenges in efficiently assembling BMC shells and encapsulating materials due to the hindering effects of sterically hindering protein domains, which prevent spontaneous formation of high-order assemblies and shells.
Innovation Solution
A fusion protein comprising BMC shell proteins with sterically hindering domains that are removable, such as Maltose Binding Protein (MBP) or Short Ubiquitin-related Modifier (SUMO), linked to the BMC shell proteins, allowing for controlled assembly by proteolytic cleavage using specific proteases like TEV or Ulp proteases, enabling the formation of structures like nanorods or shells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If sterically hindering protein domains are present on BMC shell proteins, then the proteins can be expressed and purified easily, but spontaneous assembly into high-order structures and shells is prevented
Solution Approach 1:
The sterically hindering protein domain is temporarily attached to the BMC shell protein during expression and purification, then removed by proteolytic cleavage to enable assembly. This extraction of the hindering element allows the shell protein to assemble into functional structures while maintaining ease of manufacture during the production phase.
Solution Approach 2:
The sterically hindering domain is attached beforehand to facilitate expression and purification of the BMC shell protein. This preliminary attachment enables easy manufacture, and subsequent removal triggers the assembly process, combining manufacturing ease with functional capability.
2Stability of the object's composition
If BMC shell proteins are expressed without sterically hindering domains, then spontaneous assembly occurs, but controlled assembly with temporal and kinetic control is lost
Solution Approach 1:
The sterically hindering protein domain acts as a controllable intermediary that can be attached or removed to regulate assembly. By using proteolytic cleavage of this intermediary domain, temporal and kinetic control over assembly is achieved while maintaining the spontaneous assembly capability of the shell protein.
Solution Approach 2:
The assembly state is controlled by changing the presence or absence of the sterically hindering domain through proteolytic cleavage. This parameter change (from hindered to unhindered state) enables controlled triggering of spontaneous assembly, combining adaptability with spontaneous assembly capability.
3Adaptability or versatility
If proteolytic cleavage is used to remove sterically hindering domains, then controlled assembly is achieved, but additional purification steps are required
Solution Approach 1:
The sterically hindering domain is extracted through proteolytic cleavage to enable controlled assembly. While this adds a cleavage step, the modular nature of the domain allows for systematic processing and potential automation, balancing control capability with process complexity.
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 allows for the controlled assembly of BMC shells and encapsulation of materials, enhancing encapsulation efficiency and providing a modular system for constructing composite materials and biocatalyst systems, with finer kinetic and temporal control over assembly processes.
Implementation Method 1
the sterically hindering protein domains are removable from the one or more subunits of the BMC shell protein... removable by a protease... specifically cleavable by a protease
Data Source
AI summary
The present disclosure is related to a BMC fusion protein that is capable of in vitro assembly, comprising a constituent BMC shell protein subunit and a sterically hindering protein domain that is cleavable. The BMC fusion protein is capable of in vitro assembly triggered by removal of the fused sterically hindering domain. The present disclosure is also related to a means to produce BMC shells in vitro, triggered by removal of a fused sterically hindering domain from one or more constituent BMC shell protein subunits. The BMC fusion protein enables encapsulation of broad classes of materials and biophysical studies of shell assembly, encapsulation, and permeability that would otherwise be unavailable from BMCs assembled in vivo.