BioPharm: Describe the work you're doing in the laboratory at University of Texas at Austin and what you hope to accomplish.
Stevens: In my laboratory we study ribonucleoprotein structure and function, specifically the structure and function of those ribonucleoproteins involved in gene expression. The small nuclear ribonucleoproteins (snRNPs), which are our primary interest, are present in relatively low abundance (200 to 500 molecules per cell in yeast) making their isolation and structural characterization a particular challenge. We hope to determine the low-resolution structural arrangement of these snRNPs by cryoelectron microscopy and eventually, the high-resolution structure by X-ray crystallography. To produce milligram-scale quantities of these complex molecules that contain dozens of proteins and several small RNA molecules, we require multiple kilograms of yeast. The strains we use for these experiments contain specific modifications in several genes to facilitate the snRNP purifications, so we are unable to use commercially available wild-type yeast cakes.BioPharm: What cultures do you grow in the laboratory?
Stevens: We primarily grow Saccharomyces cerevisiae in our laboratory, but we also grow Schizosaccharomyces pombe and Escherichia coli.
BioPharm: For what purpose(s) do you grow the cultures?
Stevens: For our large-scale microorganism growth, we generally use the product for protein or macromolecular complex purification. For S. cerevisiae and E. coli we perform preparative purifications of single polypeptides (E. coli), or multi-protein complexes (S. cerevisiae). For S. pombe we perform exploratory analyses of macromolecular complex composition.
BioPharm: Describe the processes and technology you originally employed to grow cultures and the progression to those you use today?
Stevens: Like most academic investigators performing similar experiments, we began experimentation using small-scale cultures of one to six liters in shake-flasks. We continue to perform small-scale experiments to troubleshoot and optimize our purification procedures. We now are able to routinely produce 400 liters of culture in a fermentor (500L Modular BioFlo Pro,New Brunswick Scientific [NBS]) in an overnight experiment. We find that although using the fermentor typically allows for faster and more dense culture growth, the procedures scale directly and the resulting product is identical to what is produced from smaller-scale cultures. We are currently attempting to optimize culture conditions with oxygen supplementation to increase the biomass yield at the end of the culture growth. With the equipment we use, there have been no challenges to overcome in scaling to this level, other than learning how to operate the equipment.
BioPharm: How customary is it for a college or university laboratory to use a fermentor to grow culture?
Stevens: In researching the purchase of the equipment for our facility, we found that many academic laboratories, departments, and colleges that used large-scale fermentation at one time have discontinued their use in the last several years. In the past decade or more, laboratories interested in milligram quantities of protein have dispensed with large-scale growth of microorganisms as bacterial over-expression systems have progressed to the point where hundreds of milligrams of product can often be purified from one to five liters of culture. In the current age of structural genomics where the low-hanging fruit of easily expressed single polypeptides will have their structures determined rapidly, these small-scale culture are ideal.