Dhinakar S. Kompala
Research Interests

Recombinant Mammalian Cell Cultures:

We have developed a novel perfusion device to selectively remove the dead mammalian cells from the high cell density bioreactor, while retaining the live, productive cells in the bioreactor. With this inclined settler device attached to a high cell density hybridoma bioreactor, we maintain high concentration of viable cells at low growth rates and more interestingly, obtain higher specific (per cell) production of monoclonal antibody. Applying the same perfusion technique to recombinant mammalian cells, we find that the specific production of a heterologous protein is lower, even though the viable cell concentration is maintained very high. Mathematical modeling of these different responses in specific productivity suggests that the protein synthesis occurs at different phases of the mammalian cell cycle for these two cell types. We have confirmed this prediction by analyzing the expression of an intracellular enzyme from commonly used SV40 and CMV promoters, using a dual-laser flow cytometer, and found the foreign gene expressed primarily in the DNA synthesis (S) phase of the cell cycle. With our prediction that a promoter with G1-phase expression of foreign gene will result in higher specific production rates, we are investigating the cell cycle phase in which some suspected G1-phase promoter systems are most active, and molecular approaches to making these promoters much stronger than their native versions. The recombinant CHO cells transfected with such expression systems in high cell density perfusion cultures to investigate whether these cells produce the desired proteins at higher specific production levels during the low growth rates.

Metabolic Pathway Engineering:

The goal of this research is to maximize the biological production and yield of specialty chemicals by incorporating new enzymatic pathways into common microorganisms, such as the brewer' s yeast. As the newly incorporated enzymes are needed only for their specific catalytic activities, it is expected the production rate and yield of end-products will be greater when the new enzymes are synthesized at only catalytic levels, and only when their substrates are made available from other reactions. This is in contrast to the first generation applications of genetic engineering, where proteins are overexpressed all the time by the use of strong constitute promoters driving their expression. Our research utilizes two families of native yeast promoters and their mutants, which are sensitive to dissolved oxygen concentration in opposite directions, for metabolic engineering of new pathways in yeast. With these newly available tools, it is possible to increase and decrease the expression of different catalytic enzymes by manipulating the extracellular dissolved oxygen concentrations. The optimal enzyme expression profiles will be predicted from cybernetic optimization procedures, and implemented in vivo by manipulating the dissolved oxygen concentration in a computer-interfaced bioreactor.

Tissue Engineering/Artificial Organs:

One recent project under this general theme involved expanding hematopoietic progenitor cells from the murine bone marrow in a well-controlled bioreactor designed to mimic the physical characteristics of the in vivo environment. A new project on this theme will address the genetic engineering of suitable host cells to respond to varying glucose concentrations by producing and secreting appropriate amounts of insulin for use in artificial pancreatic devices.