The Escherichia coli (E. coli) outer membrane protease OmpT is an
endopeptidase of the omptin family in gram negative bacteria. OmpT cleave
preferentially between two consecutive basic residues, especially Arg-Arg, and it has
been classified as an aspartyl protease based on its crystal structure although biochemical
confirmation of a catalytic aspartyl residue is lacking (Vandeputte-Rutten, et al., 2001).
Our lab has successfully engineered the P1 and P1’ specificity and selectivity of OmpT
by employing novel strategies for the isolation of enzyme variants that cleave desired
substrates from large combinatorial libraries screened by flow cytometry. However, the
engineering of proteases with altered specificity beyond the P1 and P1’ residues of the
substrate have not been demonstrated. By applying high throughput screening of large
libraries of OmpT constructed by structure-guided saturation mutagenesis of the S2
subsite (which recognizes the P2 residue), as well as random mutagenesis by error prone
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PCR and DNA shuffling, we engineered an OmpT variant exhibiting about 56 fold
change in the selectivity for the P2 position in peptide substrates. Specifically, this
enzyme preferred an acidic residue (Glu) over Tyr which is preferred by the wild type
OmpT. Molecular modeling was then employed to provide insights on how mutations
in OmpT mediated this change in P2 specificity.
A long term goal of protease engineering is to generate highly specific enzyme
variants that can be used for the irreversible inactivation of disease targets. The
anaphylatoxin C3a is a key mediator in inflammation and has been implicated with
multiple inflammatory diseases. Since the site of anaphylatoxin C3a recognized by
cellular receptors lie in its C-terminus, a protease cleaving the C-terminus of C3a could
be therapeutically relevant. Using high throughput screening and directed evolution we
successful isolated C3a cleaving enzyme variants and have characterized them
biochemically.
Finally as part of this dissertation we have employed high throughput screening
methods to dissect the substrate specificity of members of the kallikrein family of
mammalian proteases which are implicated in a number of physiological and disease
functions. The human tissue kallikrein (KLK) family contains 15 secreted serine
proteases that are expressed in a wide range of tissues and have been implicated in
different physiological functions and disease states. Of these, KLK1 has been shown to
be involved in the regulation of multiple physiological processes such as blood pressure,
smooth muscle contraction and vascular cell growth. KLK6 is over-expressed in breast
and ovarian cancer tissues and has been shown to cleave peptides derived from human
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myelin protein and the Aβamyloid peptide in vitro. Here we analyzed the substrate
specificity of KLK1 and KLK6 by substrate phage-display using a random octapeptide
library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both
trypsin-and chymotrypsin-like selectivities with Tyr/Arg preferred at the P1 site, Ser/Arg
strongly preferred at P1’ and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with
the P1 position occupied only by Arg and a strong preference for Ser in P1’. Docking
simulations of consensus peptide substrates was used to infer possible identities of the
enzyme residues that are responsible for substrate binding. Bioinformatic analysis
suggested several putative KLK6 protein substrates such as ionotropic glutamate receptor
(GluR) and synphilin. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/ETD-UT-2010-12-2353 |
| Date | 08 February 2011 |
| Creators | Li, Haixin |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
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