PharmTech: Can you describe how the process of protein expression has improved or changed over the past 10 years or so? What are one or two key technological advances that have made these changes possible?
Squires: New methods to more rapidly and reliably express proteins have proliferated over the past decade and a half. Innovations in this area have included the improvement of expression hosts to introduce more phenotypic capability to produce active, undegraded protein. For instance, hosts have been modified to overexpress folding modulators or mutated to eliminate proteolytic activities. Also, there has been a large increase in the number of these tools available commercially, putting many effective approaches within the reach of researchers with limited resources. The past several years have also seen the emergence of proprietary platform technologies, each of which offers some advantages in expressing difficult proteins or which delivers an advantageous downstream process. Examples include platforms such as a strain of Escherishia coli that secretes product to the extracellular space (Wacker Chemie AG), a toolbox of regulated promoters in the yeast Pichia pastoris (VTU Technologies), and a completely unique Pseudomonas fluorescens-based expression platform (Pfenex). The Pfenex platform has proven highly effective in expressing a variety of types of proteins and employs a high-throughput parallel processing technology in which a thousand or more unique expression strains are constructed and analyzed in about a month.Similar types of improvements have been made in the screening of mammalian expression hosts for the extent of target protein expression, including fluorescence-activated cell sorting for the selection of productive clones. These technologies are particularly effective in the identification of cell lines expressing high levels of engineered monoclonal antibodies.
In addition to these types of improvements to host and expression strategy, advances in gene design that go beyond the traditional adherence to codon usage tables have been implemented, such as the technology developed by DNA 2.0. Expression data-taught, host-specific algorithms that modulate DNA sequences while keeping protein amino acid sequence constant may add yet another dimension to the list of effective protein production tools. The community of researchers involved in all aspects of protein production is certainly much better off than it was 20 years ago in its ability to provide solutions to protein expression, but it will take much more work understanding all aspects of the nature of protein expression to arrive at universally applicable principles. Those solutions will necessarily involve an ever widening toolbox of approaches that will need to be accessed through more advanced parallel processing tools. The principle difference now is that a multidimensional approach to gene and protein expression is available to the great majority of researchers, whereas 20 years ago the process was one dimensional and digital, giving only a yes or no result.