PNNL-13501 - Pacific Northwest National Laboratory

Development of the Recombinant Protein Expression Laboratory
Study Control Number: PN99022/1350
Eric J. Ackerman, Karen Wahl, John Wahl
The post-genomic sequencing era will involve understanding proteins. This effort will require production of active
proteins in sufficient quantity for structural and functional studies. This project seeks to develop the capability for
producing recombinant proteins that are of particular relevance for the Biomolecular Networks initiative (a) . Furthermore,
this project developed methods to analyze protein activity using fluorescence methods as well as to map flexible or
unstructured regions of proteins.
Project Description
Creating libraries of protein expression vectors, proteins,
and antibodies will be valuable national assets. Producing
milligram quantities of nearly all proteins in active form
from any desired species whose genome sequence is
available is the goal of this project. The protein factory
will also produce affinity-purified antibodies of high
specificity. Each individual protein successfully
expressed by the protein factory represents potential new
research and funding opportunities for determining the
structures and functions of important biomolecules. The
collection of proteins expressed by the protein factory
represents a unique research opportunity to study
interactions amongst a library of proteins.
Cell-signaling research within the Biomolecular Networks
initiative relies on the availability of active proteins and
specific antibodies to support molecular understanding of
environmental stress responses and several non-signaling
projects in structural biology, nanotechnology,
biomaterials, and health products. Sufficient proteins and
antibodies will be available to construct large arrays for
high throughput identification of interactions and
functional assignment. Initial goals are production of
modest numbers of critical proteins and antibodies
involved in environmental cell signaling. These reagents
will hasten development of separation methodologies
enabling sensitive mass spectrometry analysis of a
diagnostic subset of the proteome responding to
environmental stress.
Introduction
In the absence of proteins, the experiments to explain
biological phenomena are often indirect and do not yield
(a) Formerly the Environmental Health initiative
64 FY 2000 Laboratory Directed Research and Development Annual Report
sufficient data for reliable modeling. Once proteins are
available, important mechanistic understandings will
emerge leading to predictions requiring even more
proteins, thus driving further discoveries. Non-proteinbased
research approaches will continue to yield
important contributions, but it is only now feasible to
attack biological problems from a comprehensive,
protein-based approach by the following generalized
strategy. Protein preparation requires obtaining the
protein-coding genes, placing genes in expression vectors,
expressing the protein, and purifying the active protein.
The functions of most proteins are unknown; therefore,
their activity cannot be measured. Nonetheless, if a
protein is expressed and purified in soluble form at high
concentrations, it will more than likely be active. The
above description is equally valid for individual proteins
or groups of proteins. This project seeks to initially
purify proteins that are crucial in cell-signaling events
related to stress responses, DNA repair-related signaling,
and apoptosis.
Approach
The initial aim of this research is to optimize our
expression/purification efforts by making proteins useful
for many aspects of environmental signaling problems. It
is essential to identify appropriate expression systems and
vectors for expression in E. coli, pichia, and insect cells.
Some proteins can be made only by expression in
mammalian cells. Nonetheless, our initial efforts are
limited to non-mammalian expression systems because
1) retrofitting our existing building with the appropriate
filtration, water, air-locks, etc., is not cost-effective;
2) many interesting proteins can be expressed in
microbes, thus avoiding the need for mammalian