Qiagen Interview with Dr. Stefan Kubick (RiNA GmbH) about exciting new developments in membrane protein research
* RiNA and QIAGEN cooperate in the development and commercialization of new solutions in the field of cell-free protein expression and labeling.
What are your research interests?
The key area of our research activities at RiNA GmbH is the development of novel cell-free protein synthesis systems, also termed in vitro translation systems. We have commercialized several in vitro protein expression systems based on highly productive prokaryotic and eukaryotic lysates containing all essential factors for transcription and translation. Cell-free protein synthesis is a valuable tool for understanding how mRNAs are translated into functional polypeptides and is a powerful technique for the production of a wide variety of proteins, including membrane proteins and glycoproteins.
We have developed a technique for the standardized production of translationally active eukaryotic lysates from insect cells. In contrast to other cell-free protein synthesis systems, e.g., rabbit reticulocyte lysates and wheat germ extracts, our homogenization procedure avoids any serious breakdown of membrane vesicles already existing in the cytoplasm of the prepared eukaryotic cells. We have demonstrated the functional integrity of these subcellular components by showing signal peptide cleavage as well as glycosylation of in vitro expressed membrane proteins. Our cell-free protein synthesis systems offer the unique opportunity to use the highly efficient translational machinery while introducing novel functional groups at specific sites in proteins.
Can you tell us more about your work at the university?
The work at the university involves lectures, tutorials, and practical courses. Lectures mainly focus on theoretical aspects of membranology such as membrane lipid functions, membrane protein synthesis, as well as sorting, folding, and targeting of transmembraneous and membrane-attached proteins. The practical courses are designed to familiarize students with current membrane protein research and to expand on the protein expression systems presented in the previous lectures. Topics include the synthesis and fluorescent labeling of various structurally divergent transmembraneous proteins in cell-free systems, screening of their solubility, and analysis of posttranslational modifications, e.g., glycosylation.
Why are membrane proteins so fascinating for you?
Membrane proteins represent approximately 30% of the total proteins from an organism and an increasing number of membrane protein sequences without attributed function are constantly discovered in various genome sequencing projects. Integral membrane proteins are involved in essential biochemical processes as they perform critical roles in the cell cycle by regulating signalling, metabolism, transport, and recognition. Dysregulation of their biological activity in response to bacterial or viral infection, as well as in cancer often leads to severe diseases. Considering that these proteins play a central role in drug discovery as potential pharmaceutical targets, it is now imperative to go deeper into their structure to understand their molecular and physiological function.
What do you see as greatest challenge in membrane protein research in the next few years?
The molecular analysis of membrane proteins lags far behind that of cytosolic soluble proteins. Membrane proteins are highly underrepresented in structural data banks. One of the major difficulties in the study of membrane proteins is to recover suitable amounts of recombinant proteins in correctly folded structures, displaying proper functions and activities. Cotranslational integration of membrane proteins into an appropriate lipid bilayer constitutes a critical step often necessary for obtaining native-like and functionally active membrane proteins. A key step in the biosynthesis of membrane proteins is their complete translocation across the eukaryotic endoplasmic reticulum membrane. Unfortunately, membrane integration of recombinant membrane proteins upon their production in conventional cell culture systems of bacterial, yeast, mammalian, or insect origin often affects the integrity of the cellular membranes resulting in growth retardation or even lysis of the host cells. Blocking cellular transport and posttranslational processing systems by overproduced heterologous membrane proteins may cause further toxic effects. Examples of successfully overproduced membrane proteins in preparative amounts are relatively rare and even frequently associated with the aggregation and inactivation of the recombinant membrane protein by inclusion body formation or proteolytic degradation.
Any attempt to develop a new method for expressing membrane proteins must consider these challenging problems. In this context, cell-free expression is a powerful, flexible, and expanding technology. The ability to easily adapt the reaction conditions for each individual membrane protein and the wide possibilities of product modifications provides new opportunities. In the postgenomic era, cell-free protein synthesis has the potential to become one of the most important high-throughput technologies for functional genomics and proteomics. The rapidly accumulating information obtained from these high-throughput approaches and systematic reaction condition screens will provide a comprehensive and reliable database of knowledge for the preparative-scale cell-free synthesis of many membrane proteins.
When you are doing your “hands on training” at the university, what is your advice for the students starting in the field of membrane proteins?
The aim of the “hands on training” at the university is threefold: the first is to review, assess, and share information on the most recent scientific and technological advances in the field of membrane protein expression. The second is to create the opportunity for scientists from different parts of the world to discuss and share their research findings and to explore the possibilities of cell-free protein synthesis. The third objective is to support young researchers in different areas of biochemistry and molecular biology, in particular applied research, to favor the development of a multidisciplinary approach to the study of membrane proteins.
My personal advice for the students starting in the field of membrane proteins is to extensively monitor the available expression systems and their suitability for the individual membrane protein they wish to analyze before starting any experiment.