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Trenzyme – optimal expression of recombinant proteins

Trenzyme GmbH in Konstanz offers services and products in the fields of genomics and proteomics. The company specialises in contract research and custom R&D services and is also working on new and improved products and processes. The company recently developed a new method for the rapid identification of optimal expression conditions for recombinant proteins.

“Recombinant proteins are of central importance in the life sciences,” said Dr. Reinhold Horlacher, founder and CEO of Trenzyme GmbH, relating to the company’s major business focus. Recombinant proteins – therapeutic antibodies for example - are used in the pharmaceutical industry as active compounds for the treatment of disease. In addition, they also play a pivotal role in pharmaceutical R&D. Industrial biotechnology uses enzymes for the conversion of substances. Large amounts of recombinant proteins are required to do this, and such quantities can only be produced using effective, high-quality processes. Trenzyme therefore relies on a proprietary method that identifies the expression conditions that are best suited for the production of sought-after recombinant proteins.

A building block system for finding many answers

Dr. Reinhold Horlacher, founder and CEO of Trenzyme GmbH, works on the optimisation of expression conditions for recombinant proteins. © Trenzyme GmbH

The Konstanz-based company uses a modular ‘building block’ system, which can deal with a specific question in a number of different ways. The modules are composed of different elements at different levels. First, the host organism for the expression of a protein of interest is selected from a collection of E. coli strains, yeasts, and insect and mammalian cells. This decision then defines the range of suitable expression vectors, as the vectors are adapted to either bacterial, yeast, insect or mammalian hosts. The final vector selection is made on the basis of the target protein’s properties, e.g. the secreted or cytoplasmic expression in mammalian cells, inducibility of the protein or the coupling to affinity markers. 

“A broad range of vectors is available for bacterial systems, including vectors with different promoters, resistance markers and different copy numbers,” said Horlacher. “In addition, all of the 70 or more vectors that we have at our disposal have the same general structure, so any target sequence can be rapidly inserted into any of the vectors in one step,  without time-consuming modification steps,” Horlacher continued. The selection of useful combinations is also based on past experience and the specific objective. “If the protein of interest has glycosylated residues, E. coli cannot be used as host since bacteria do not glycosylate proteins," says Horlacher. 

This open-ended approach consists of several steps. First, small amounts of the protein are expressed in a maximum number of useful variations with the aim of qualitatively and quantitatively identifying optimal conditions. Second, the chosen conditions are transferred to the large scale and further optimised. “Key in this approach is an excellent readout of protein quality, which is synonymous with protein activity,” Horlacher explained. At Trenzyme, everyday laboratory practice clearly shows the importance of this additional test: for example, a protein first tested in E. coli and in mammalian cells with different vector constructs and subsequently tested for enzyme activity was expressed in both systems, including on the larger scale in E. coli. “However, the activity test showed that a functional enzyme was only synthesised in mammalian cells, so that E. coli could not in the end be used as host for this protein,” says Horlacher. Trenzyme GmbH also supports companies that lack methods for assessing protein quality in the establishment of suitable assays.

Homogeneous cell lines using the ExoIN technology

A micro-bioreactor is used to select host-vector combinations with the highest protein expression rate and protein activity from a broad range of different combinations. © Trenzyme GmbH

The modularity and range of the system is made possible by special technical subtleties. “We have incorporated some new elements into our system; the ExoIn technology is one such example,” said Horlacher. ExoIn is Trenzyme GmbH’s proprietary method for the generation of homogeneously expressed, stable cell lines. The ExoIN vector system couples target protein synthesis with the synthesis of a selection marker (e.g. antibiotics resistance). In contrast to many other systems, coupling is done on the protein level, so that both proteins are translated together. It can be safely assumed that the cells express the target protein when the marker protein is also present after transfection; if the cells manage to grow under selective conditions (antibiotics), they then synthesise the target protein; negative cells die. “In addition to the incredible speed, another big advantage of the technology is the possibility to specifically select different expression levels. No other system does this, and this is invaluable, primarily with regard to functional issues,” Horlacher explained.

Cell-line development as a mainstay of the future

Trenzyme GmbH was spun out from the University of Konstanz in 2000 and has since grown continuously. The company started off with a team of two young scientists, and now has a staff of 14. Moreover, it received DIN EN ISO 9001:2008 certification in 2009. An electronic quality management system for flawless process documentation and efficient corporate management guarantees Trenzyme’s customers transparency of all processes. 

In 2007, the company added the development and production of stable mammalian cell lines to its core area, i.e. the production of recombinant proteins. “As development partner to global pharmaceutical and biotechnology companies, we have since developed more than 300 stable cell lines in which a desired protein is expressed or silenced,” says Horlacher. The new developments led to the foundation of Cytobox in 2009, a Trenzyme subsidiary that specialises in the production of cell lines. The range of Trenzyme services includes full-service DNA work (‘gene to plasmid’), the production of recombinant proteins (‘gene to protein’) and cell line development (‘gene to cell line’), along with the broad range of molecular biology, protein biochemistry and cell culture methods required for plasmid, protein and cell line production. 

In the coming years, Trenzyme GmbH will continue to develop new methods and applications, primarily in the field of cell culture. “At the moment we are working on a technology that enables the production and cryostorage of transfection-competent cell lines, including primary cells,” said Horlacher. This technology was also licensed in November 2011 to Lonza Group Ltd., a leading provider of products and services to the pharmaceutical, healthcare and life sciences industries.

Further information
Trenzyme GmbH
Dr. Reinhold Horlacher
Byk-Gulden-Str. 2
78467 Konstanz
Tel.: +49 (0)7531/ 12 29 00
Fax: +49 (0)7531/ 122 90 11
E-mail: reinhold.horlacher(at)trenzyme.com

Website address: https://www.biooekonomie-bw.de/en/articles/news/trenzyme-optimal-expression-of-recombinant-proteins