Description

The collection described herein consists of parts, necessary to build a novel liposome-based platform for membrane protein (MP) synthesis, integration, and display. It is comprised of a cellular MP integration machinery, target membrane proteins and a unique chaperone. Guide RNAs introduce genetic modifications to nascent ribosome proteins for purification and further ribosome anchoring to nickel-chelating lipids of liposome membrane. Synthetic RNA thermometers act as a user-controllable mechanism for regulation of target protein expression and high-yield synthesis in an inducible temperature range. Thermoswitches elegantly complement the function of BAM complex which facilitates membrane-assembly of beta-barrel bearing proteins. Our target membrane proteins are fused with Mistic protein for a superior integration into the membrane. Additionally, MPs of our choice expose designated antibodies on the surface of the liposome. While all with different functions, together these parts establish a unique level of complexity and create a universal synthetic system for membrane protein research in liposomes.

Cellular MP integration machinery, the BAM complex, consists of five essential subunits, hence parts. They are well characterized and maintain their identical cellular functions when encapsulated inside liposomes. Parts are already labeled with His-tag, to ease the purification process of BamA-E proteins using the His-tag affinity chromatography.

Previous parts of BAM complex are essential for viable membrane insertion of beta-barrel bearing membrane proteins. There are two different membrane proteins in our collection to suit the needs of a user. OmpA is a member of OMP superfamily and one of the most well described and characterized proteins of gram-negative bacteria outer membrane. In this collection we use well characterized Lpp-OmpA (46-159) fusion protein. Another membrane protein in our collection is the beta-domain of Iga protease, which is attractive due to its autotransporter and pore-forming properties.

Our part collection also contains 11 different synthetic RNA thermometers. These parts act as thermosensors that regulate gene expression by temperature-induced changes in RNA conformation. Thermoswitches are the most universal feature of our part collection, as they can be easily applied not only in our system, but also any other in vivo or especially in vitro system that needs to incorporate chemical-free gene regulation. The applications of these parts range from RNA-ordered gene expression dynamics to tightly controlled modularization of genetic cascades. In this collection, however, RNA thermometers are needed to lock the initial translation of our target constructs bearing plasmids. It is needed to give enough time for BamA protein to correctly fold and insert into liposome membrane where it can accept and assemble our target beta-barrel bearing membrane proteins. It is one of many examples demonstrating the beautiful interplay between our parts.

A unique feature of this collection is Mistic. It is a fresh and still understudied protein, whose mechanism of action is unclear. However, for what is known, Mistic possesses functional ability to chaperone membrane proteins when fused to their N-terminus to membrane by associating with it. Its function is autonomous meaning not Sec-dependent. All these features make Mistic a perfect piece for our composite parts also containing membrane proteins and antibodies.

The part collection further features a camelid class antibody called GFP Nanobody and a single chain variable fragment (scFv) of anti-vaginolysin. These parts serve to prove the versatility and operation of our system. Comprising composite constructs, both GFP Nanobody and anti-vaginolysin can be displayed on liposome surface by membrane proteins which are fused both to the antibodies and protein Mistic. These parts prove that our system can display a full range of molecules: versatile antibodies, peptides, and other proteins, thus becoming a universal and smart novel exposition system.

Final feature of our part collection is parts bearing the sequences of guide RNAs for specific nascent ribosome protein modifications. It is extremely needed to ensure that an in vitro protein expression system will be high-yielding. We introduce a possibility to modify nascent ribosome proteins L23 and L24 by labelling their C’ terminal ends with a his-tag allowing ribosome anchoring to lipid membranes via an interaction between a his-tag and metal chelating lipids. Third part of this type has a sequence of gRNA that introduces a Strep-tag to the C’ terminus of protein L12 allowing to purify engineered ribosomes using Strep-tag affinity chromatography.

Although various and very different, when used right these parts elegantly interplay and orchestrate the assembly of novel synthetic membrane protein synthesis, display, and research system in liposomes.