Douglas S. Clark - US grants

This research involves novel strategies to produce antibody polypeptides with optimal structures for immobilization to insoluble carriers. Each approach is designed to result in immobilized, monovalent antibody fragments with their combining sites facing out from the carrier surface, and attachment to the carrier will be achieved in a highly specific manner through a site on the polypeptide far removed from the combining site itself. Attachment in this fashion should allow complete retention of antibody binding activity and thus lead to improved techniques of affinity purification. In addition, immobilizing antibody fragments rather than the much large whole antibody should increase the number of antibody combining sites that can be immobilized on the carrier surface. In the case of Fv fragments, site-directed mutagenesis will be used to incorporate a single attachment site at a desired location on the polypeptide. The modified polypeptide will then be expressed in E. coli. Production of Fv fragments in E. coli may prove to be a superior alternative to present-day hybridoma technology, which is frequently the limiting factor in monoclonal antibody production and isolation. Moreover, both site- directed mutagenesis and random mutagenesis can be used to rapidly obtain antibody polypeptides with variant binding activities. Protein engineering may also be used to introduce pH or metal-regulated binding affinity into the combining site.

This collaborative project between workers at Berkeley and UCSF is designed to produce new polymeric peptides with useful properties. The investigators are all excellent scientists, although all well supported by other grants. In particular Dr. Craik has another NSF grant which duplicates his effort in this proposal. The P.I. has developed enzymatic methods for reacting amino acid esters in organic solvents with free amino acids to generate dipeptides. The approach has been extended to a tetra peptide, but the strategy seems unlikely to produce useful polymers, although it might be practical for short peptides. It should be noted that carboxypeptidase cannot be employed in such syntheses because it does not have and acyl enzyme intermediate. There are some interesting ideas in this proposal, but the practicality of synthesizing polypeptides by these approaches seems minimal.

The University of California Berkeley will receive ARI Facilities support to create modern facilities for researchers working in a unique, integrated Program in Biological Chemistry and Engineering within the College of Chemistry. The renovated facilities will promote a strong interaction among bioinorganic chemists, biochemical engineers, and bioorganic chemists working in the areas of biotechnology and environmental research. The renovations activity funded by this award will be directed at the improvement of 1,305 square meters in 30-year old Latimer Hall and will consolidate researchers. Program activity will be enhanced as researchers are currently located in geographically distributed campus laboratories that are between 32 and 77 years old. A 7 month- long space assessment precedes the project which will involve gutting much of the interior space, installation of modular laboratory units and new fume hoods and upgrades to the mechanical, electrical and plumbing services for increased safety and improved efficiency. Provision of adequate facilities will enable the basic discovery processes to be linked more closely with the development process. Biochemical engineers will study separation techniques for the recovery of biological products, biomimetic adsorbents for metal removal and recovery and new approaches to bioremediation. Bioinorganic chemists will study metal ion transport that is essential to life processes, the use of chelating agents in the sequestration of heavy metals, and lanthanide complexes for possible use in enhancing MRI. A total of 8 professors and 117 graduate students and postdoctoral fellows will benefit from these improvements.

9421862 Clark This project is on research to study the production of enzymes from extremophiles by expression of these enzymes in mesophiles, using recombinant DNA technology. The objective is to make available large quantities of these enzymes for biophysical and biochemical characterization and ultimately for enzymatic applications, without the need for culturing extremophiles directly. The specific objectives of this research include: (1) to clone and identify gene sequences from the hyperthermophile Pyrococcus furiosus and related thermophiles; (2) to express genes in heterologous systems at high pressure, and characterize recombinant enzymes under elevated pressure and temperature to establish optimal specific activity and assembly; and (3) to develop bioprocessing methods for obtaining enzymes in sufficient quantity for structural determination and biotechnological applications. ***

9816490 Clark Hyperthermophilic microorganisms may be suitable models for ancestral, and possibly extraterrestrial, biosystems because their extraordinary adaptive capabilities allow them to colonize a wide range of subsurface volcanic areas on Earth. Deep sea hydrothermal vents and terrestrial hot springs support highly diverse ecosystems without access to solar energy, harboring autotrophic microorganisms and their dependant heterotrophic micro and macro communities in geochemical conditions similar to those that may exist below the surface of Mars or Europa. Hyperthermophiles also dominate most of the deeper branches of the universal phylogenetic tree, suggesting that ancestral microorganisms may have been thermophilic. The environmental limits for growth and survival of known hyperthermophilic species, as well as newly isolated strains will be established, using a combination of bioengineering, microbiology and molecular biology. This collaborative project with Dr. Frank T. Robb of the University of Maryland, Baltimore (Award MCB-9809352) emphasizes molecular adaptations to high pressure and high temperature, with the following objectives: 1. To determine the effects of supraoptimal temperatures and pressures on the survival and growth rates of existing hyperthermophilic microorganisms; 2. To utilize pressurized continuous fermentation systems for isolation and culture of new hyperthermophilic microbial strains and; 3. To examine physiological adaptations and genetic regulation of gene expression in response to transient challenges by heat and high pressure. The hypothesis being tested is that hydrostatic pressure may greatly extend the upper temperature limits of growth and survival of hyperthermophiles. Ongoing studies by these researchers have established that many enzymes from thermophiles display enhanced thermostability under pressure. Further, the growth rate of Methanococcus jannaschii accelerated five-fold and its maximum temperature for methane production rose by 6 degrees C in response to pressure, and the growth rate and ATP production of a newly characterized abyssal hyperthermophile, Pyrococcus horikoshii, were elevated under pressure. Novel equipment for incubating hyperthermophiles in continuous culture, under pressure and with thermal cycling, will be combined with molecular biology to explore the adaptive responses of hyperthermophiles in extremis. Gene expression, membrane lipid composition and morphology will be examined. The genes that are induced under conditions approaching lethality, will be identified by subtractive cloning and transcriptional assays. Upper survival limits of the abyssal hyperthermophiles as a function of pressure, using isolates from a shallow terrestrial sampling site to provide control data for pressure responses, will be determined. Enrichment cultures of hyperthermophiles from the vent systems of the back-arc region of the Northwest Pacific (Okinawa Trough and Uzzon Caldera on the Kamchatka Peninsula) will be incubated at temperatures and pressures exceeding those tolerated by known strains, thus using survival rather than rapid growth as the criterion for selection of new isolates. The phylogenetic positions and physiological requirements of the new strains will be determined. ***

This proposal pursues an older observation that the lyophilization of certain enzyme catalysts in the presence of highly ionic, non-buffering salts, e.g., KCl, can increase the catalytic rate in non-aqueous systems by 3-4 orders of magnitude. This has been demonstrated primarily on subtilisin. The specific aims of the original proposal are:
To elucidate the mechanism(s) of enzyme activation in organic solvents with salts
To extend the concept to a broad range of enzymes and reactions. To utilize the activated enzymes for combinatorial biocatalysis. In the revised and funded proposal, Aim 3 has been dropped and a more detailed examination of the mechanism and scope of the phenomenon has been added.

The Biochemical Engineering Conference serves as the premier meeting for the Biochemical Engineering community. The focus of this Conference will include important core areas of Biochemical Engineering (fermentation/cell culture and downstream process development) as well as exciting new frontiers of Biochemical Engineering (nanotechnologies, the "omics" of proteomics, genomics and physiomics, marine and environmental biotechnology). The Conference will illustrate how the same engineering fundamentals (unit operations, mass and energy transport, thermodynamics, and kinetics) that form the basis of traditional areas of Biochemical Engineering are being applied to drive advances in both areas. In addition to the regular sessions, two workshops are planned. A workshop entitled "Biochemical Engineering Education: fundamentals versus state-of-the-art" will focus on the often-competing needs to incorporate engineering fundamental into the curriculum as well as state-of-the-art applications. This workshop will feature representatives from industry and academe. A second workshop entitled "New Directions in Biochemical Engineering Research" will focus on potentially new areas for research in Biochemical Engineering. The discussion will have as panelists both academic and industrial representatives. Each panelist will be asked to prepare a single transparency and speak for no longer than 5 minutes. Following the presentations, input from the audience will be requested.

The objective of this project is to improve recombinant expression systems. The major goals are to discover and characterize pathways that facilitate protein folding and subunit assembly in hyperthermophiles. The components of these pathways will be inserted into recombinant host cells to preempt induction of stress responses during recombinant expression, and to enhance the stabilization and solubilization of recombinant protein. Building on the approach and findings of the investigators previous research, the specific objectives are: (1) to determine the functions of interacting proteins encoded by the 23 genes in the heat shock regulon of the hyperthermophile, Pyrococcus furiosus, (2) to identify functional complexes of thermostable heat shock proteins and express them in E. coli, yeast and mammalian cells to enhance the durability of these cells, and (3) to determine the effects of combinations of chaperonins on high level recombinant expression of proteins in intact cells as well as cell-free transcription-translation systems.

The overall goals of this research are to achieve rate enhancements of several thousand fold, and to elucidate the underlying mechanism(s) for the activation process. This research will therefore provide new insights into the factors that govern enzyme activity in organic solvents, and will enable enzymes to play an expanded role in industrial processes, including chemicals and pharmaceuticals synthesis, polymer synthesis, drug discovery, and waste treatment, among other emerging applications of biocatalysis. Moreover, the same techniques that lead to improved enzyme function in organic solvents are likely to be of use in the stabilization of dehydrated protein systems, which is a critical issue in biotherapeutic protein formulation and delivery. Thus, this work has broad impact, ranging from biocatalysis to drug discovery to biopharmaceutical formulations.

Microbes can make organic molecules with complex structures. Many of these molecules or their analogs are central ingredients in a variety of products, but the diversity of these molecules is limited by the range of the reactions catalyzed by natural enzymes. Engineering cells to produce artificial metalloenzymes (AMEs) will expand the range of possible reactions. Novel biosynthetic pathways will be created by complementing natural enzymes with AMEs. Graduate students and postdoctoral associates will be trained in this convergent field of synthetic chemistry and synthetic biology. Outreach activities to encompass topics related to artificial biosynthesis will be offered to underserved high school students and teachers, as well as to K-8 students.


This project will build upon the general concept of creating artificial biosynthetic pathways containing artificial metalloenzymes and preliminary results showing the feasibility of creating these pathways in bacteria. We will increase the numbers and types of microorganisms that can host the chemistry catalyzed by artificial metalloenzymes to expand the range of natural products that react with AMEs; expand the types of metallo-cofactors that are incorporated intracellularly into AMEs to increase the scope of unnatural reactions in these pathways; and combine the abiotic chemistry with natural biosynthesis in varying sequences. Specifically, we will 1) introduce AMEs into Streptomyces strains and test activity on heterologously produced terpenes and polyketides; 2) incorporate new cofactors into AMEs expressed in E. coli and Streptomyces; 3) broaden the scope of transformations catalyzed by AMEs in the artificial biosynthetic pathways to encompass abiotic C-H bond functionalizations; 4) create pathways in which the unnatural chemistry occurs in the middle of the artificial biosynthesis; and 5) elucidate the pathways for diazo-containing small molecules. By doing so, we will generate the fundamental knowledge and demonstrate guiding principles to create artificial biosynthetic pathways that convert simple carbon sources to valuable unnatural products in whole microorganisms.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.