Proteins are the basic building blocks of cells. They tend to be biologically active in combination with many other proteins rather than as isolated entities. Such protein complexes play a key role in many vital processes in our bodies. In the field of medicine, proteins are used, amongst other things, as antibodies for developing vaccines. One of today’s greatest challenges is to develop effective vaccines against cancer. An international team of researchers, including Professor Stefan Bräse and his team from the Karlsruhe Institute of Technology (KIT), has developed an innovative platform called MultiBacTAG that can be used to engineer recombinant proteins with custom functionalities for biotechnological and pharmaceutical applications.
All living organisms contain DNA in their cells. DNA carries all the genetic instructions required for an organism’s proper development and functioning. While certain DNA sections contain the instructions for making specific proteins, protein complexes are the “machinery” that actually translates the genetic information stored in DNA into proteins. The resulting proteins have a variety of functions: they catalyse important cellular processes and transport substances, pump ions across membranes, recognise signalling molecules and degrade or convert substances. A particularly important function of protein complexes is to build new cells and repair existing ones.
The entire set of protein molecules present in a particular cell at a given time is called a proteome. In the proteome, a constant equilibrium is maintained between protein synthesis and degradation. The composition of the proteome is thus continually changing. These changes are controlled by complex regulatory procedures that can be interfered with by active pharmaceutical ingredients and drugs. "This opens up exciting perspectives for medicine and the life sciences", says Prof. Bräse from the Institute of Organic Chemistry at KIT, who is involved in intensive international research in applied chemistry and has received prestigious awards such as the ORCHEM Prize for Young Scientists and the Richard Zsigmondy Prize.
Cells use protein complexes to “read” genes and translate them into proteins. As the language of genes is universal, bacterial, yeast and mammalian cells can therefore read human genes and create human proteins. Genetic engineering tools are used to isolate, copy, amplify and introduce a human gene of interest into production cells. It is also possible to specifically modify genes or assemble them from parts of two or more genes. The resulting recombinant proteins, so-called designer proteins, can be used in research and medicine. They are the cornerstones that generate specific protein complexes such as vaccines and multiple signal cascade proteins. Bacterial, yeast, insect and mammalian cells are used as production organisms.
Although bacterial cells such as E. coli are easy to culture and able to produce high protein yields, they are unsuitable for the biosynthesis of designer proteins. Most protein-based drugs only become effective when modified, for example when they are glycoslyated, i.e. carry a sugar residue. As such complex protein complexes cannot be expressed in simple bacteria, mammalian cells are needed for this purpose.
"We have been working intensively on the MultiBac system for some years now. It is a baculovirus expression vector system that can be used for producing eukaryotic protein complexes,” says Prof. Bräse who is working alongside Dr. Edward A. Lemke, the head of a group of researchers carrying out high-resolution studies of protein plasticity at EMBL (European Molecular Biology Laboratory) in Heidelberg. Both are part of a group of international researchers. A “gene shuttle” is needed to transfer the gene carrying the information for making recombinant proteins into a cell that subsequently expresses the desired gene product in high quantities. This can now be achieved with the new MultiBacTM expression system. The system uses baculovirus gene shuttles in combination with the Sf21 insect cell expression machinery.
The MultiBac system has been specifically designed to produce eukaryotic protein complexes composed of many domains. The system, which comprises all individual steps from the introduction of the genes into a constructed baculovirus genome to protein production in insect cells and analysis of the protein using standard protocols, was developed by Imre Berger at the Swiss Federal Institute of Technology ETH (Zurich) and is available as an open access platform at the EMBL in Grenoble, France. The platform helps many researchers in industry and academia speed up research projects involving protein complexes.
Genetic code expansion (GCE) allows researchers to reprogramme a protein of interest (POI) to site-specifically incorporate a non-canonical amino acid and create proteins with novel functions. Non-canonical amino acids are amino acids that contain a side group (e.g. cyclooctene) that does not occur in nature. GCE involves introducing a stop codon (TAG) at a specific site in the coding gene of the POI. A specific tRNA/tRNA synthetase pair that specifically recognises the non-canonical amino acid subsequently expresses the corresponding protein. In order to achieve this, the tRNA/tRNA-synthetase pair that selectively recognises the non-canonical amino acid will have previously been transferred into the baculovirus DNA.
By outfitting the MultiBac system with the molecular tool kit required for genetic code expansion in eukaryotes and a specially designed tRNA/tRNA synthetase pair, the international team successfully produced in insect cells multidomain protein complexes that carry unnatural amino acids. "This opens up a variety of applications," says Bräse. The so-called MultiBacTAG system has been available since October 2016, and a paper describing how it works and its wide range of applications was published in the renowned scientific journal Nature Methods in December 2016.1 “The user-friendliness of MultiBacTAG is, first and foremost, due to the excellent documentation of the individual work steps. As the components that lead to a change in the genetic code of a POI are inserted into the backbone of MultiBacTAG, the user can directly apply our expansion system without prior experience or training in GCE,” says Bräse highlighting the platform’s advantages. The system can be obtained free of charge when used for academic purposes. Investors are asked to contact EMBLEM, the EMBL’s technology transfer organisation. “We can produce the desired proteins with built-in target functionalities in large quantities by combining the MultiBac system with site-specific genetic engineering methods, such as MultiBacTAG,” says Bräse.
The applications of the MultiBacTAG system range from the fluorescence labelling of specific target proteins (for diagnostic purposes) to the production of active pharmaceutical ingredients. The chemical biologists have already used the system to engineer Herceptin, a monoclonal antibody against breast cancer that can recognise specific breast cancer cells in human tissue. “MultiBacTAG opens up a wide range of possibilities for designing proteins used for biotechnological and pharmaceutical applications. Research into protein complexes and their functional interactions can benefit from this,” says Bräse, highlighting the exciting application possibilities for the platform.
1 Koehler C, Sauter PF, Wawryszyn M, Girona GE, Gupta K, Landry JJ, Fritz MH, Radic K, Hoffmann JE, Chen ZA, Zou J, Tan PS, Galik B, Junttila S, Stolt-Bergner P, Pruneri G, Gyenesei A, Schultz C, Biskup MB, Besir H, Benes V, Rappsilber J, Jechlinger M, Korbel JO, Berger I, Braese S, Lemke EA. (2016). Genetic code expansion for multiprotein complex engineering. Nat. Methods doi: 10.1038/nmeth.4032