6 research outputs found
Directed evolution methods for overcoming tradeâoffs between protein activity and stability
Engineered proteins are being widely developed and employed in applications ranging from enzyme catalysts to therapeutic antibodies. Directed evolution, an iterative experimental process composed of mutagenesis and library screening, is a powerful technique for enhancing existing protein activities and generating entirely new ones not observed in nature. However, the process of accumulating mutations for enhanced protein activity requires chemical and structural changes that are often destabilizing, and low protein stability is a significant barrier to achieving large enhancements in activity during multiple rounds of directed evolution. Here we highlight advances in understanding the origins of protein activity/stability tradeâoffs for two important classes of proteins (enzymes and antibodies) as well as innovative experimental and computational methods for overcoming such tradeâoffs. These advances hold great potential for improving the generation of highly active and stable proteins that are needed to address key challenges related to human health, energy and the environment.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154495/1/aic16814_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154495/2/aic16814.pd
Optimizing a bacterial sRNA scaffold for targeting multiple mRNAs, filtering off- target mRNA interactions, and balancing metabolic pathway flux
RNA is central to gene expression control in cells, and yet the small regulatory RNAs (sRNAs) of bacteria are still in the early stages of development as synthetic biology tools. The ubiquity and diversity of sRNAs in bacteria bodes well for engineering synthetic sRNA control of metabolic pathways, particularly in organisms with poorly developed genetic tools. These sRNAs regulate mRNA targets by RNA:RNA base-pairing interactions, and sRNAs have been retargeted to regulate non-native mRNAs for metabolic engineering applications.1-3 However, an ongoing concern about sRNAs as tools is their potential for hybridizing to off-target mRNAs. Here we describe the development, optimization and implementation of a structured sRNA scaffold with improved target discrimination relative to an unstructured antisense sRNA scaffold. Native DsrA sRNA4,5 contains two striking stem-loop antisense motifs that use antisense base-pairing to coordinately regulate translation of two E. coli mRNA targets. Previously6 we created a genetic system for retargeting DsrA simultaneously to two non-native mRNA targets in E. coli. Next, we expressed a retargeted E. coli sRNA variant in C. acetobutylicum cultures to improve n-butanol biofuel fermentation yield and selectivity. We achieved this goal by retargeting a DsrA sRNA variant to tune-down expression of an essential clostridial hydrogenase and increase NADH levels in the fermentation culture. Finally, we used this E. coli sRNA genetic system to demonstrate that the stem-loop antisense âfingerloopâ structures can be configured to exclude certain off-target mRNA interactions. This fingerloop antisense motif constitutes a very promising programmable targetâmRNA control element that is modular, discriminating, and portable between organisms. Since fine-tuning and balancing metabolic pathway flux is an important scale-up parameter, this nanoscale sRNA tool should be particularly useful in industrial scale bacterial fermentations of biofuels and specialty chemicals.
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Sensitive detection of glucagon aggregation using amyloid fibrilâspecific antibodies
Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (20 times more sensitive than detection using a conventional, amyloidâspecific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (20 times more sensitive than conventional methods for detecting glucagon fibrils.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150615/1/bit26994_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150615/2/bit26994.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150615/3/bit26994-sup-0001-Supporting_Information__submission_.pd
Column-Free Purification Methods for Recombinant Proteins Using Self-Cleaving Aggregating Tags
Conventional column chromatography processes to purify recombinant proteins are associated with high production costs and slow volumetric throughput at both laboratory and large scale. Non-chromatographic purifications based on selective aggregating tags have the potential to reduce costs with acceptable protein yields. A significant drawback, however, is that current proteolytic approaches for post-purification tag removal after are expensive and non-scalable. To address this problem, we have developed two non-chromatographic purification strategies that use either the elastin-like polypeptide (ELP) tag or the β-roll tag (BRT17) in combination with an engineered split intein for tag removal. The use of the split intein eliminates premature cleavage during expression and provides controlled cleavage under mild conditions after purification. These self-cleaving aggregating tags were used to efficiently purify β-lactamase (β-lac), super-folder green fluorescent protein (sfGFP), streptokinase (SK) and maltose binding protein (MBP), resulting in increased yields compared to previous ELP and BRT17-based methods. Observed yields of purified targets for both systems typically ranged from approximately 200 to 300 micrograms per milliliter of cell culture, while overall recoveries ranged from 10 to 85 percent and were highly dependent on the target protein
Retargeting a Dual-Acting sRNA for Multiple mRNA Transcript Regulation
Multitargeting small regulatory RNAs
(sRNAs) represent a potentially
useful tool for metabolic engineering applications. Natural multitargeting
sRNAs govern bacterial gene expression by binding to the translation
initiation regions of protein-coding mRNAs through base pairing. We
designed an <i>Escherichia coli</i> based genetic system
to create and assay dual-acting retargeted-sRNA variants. The variants
can be assayed for coordinate translational regulation of two alternate
mRNA leaders fused to independent reporter genes. Accordingly, we
began with the well-characterized <i>E. coli</i> native
DsrA sRNA. The merits of using DsrA include its well-characterized
separation of function into two independently folded stem-loop domains,
wherein alterations at one stem do not necessarily abolish activity
at the other stem. Expression of the sRNA and each reporter mRNA was
independently controlled by small inducer molecules, allowing precise
quantification of the regulatory effects of each sRNA:mRNA interaction <i>in vivo</i> with a microtiter plate assay. Using this system,
we semirationally designed DsrA variants screened in <i>E. coli</i> for their ability to regulate key mRNA leader sequences from the <i>Clostridium acetobutylicum n</i>-butanol synthesis pathway.
To coordinate intervention at two points in a metabolic pathway, we
created bifunctional sRNA prototypes by combining sequences from two
singly retargeted DsrA variants. This approach constitutes a platform
for designing sRNAs to specifically target arbitrary mRNA transcript
sequences, and thus provides a generalizable tool for retargeting
and characterizing multitarget sRNAs for metabolic engineering
Small-Molecule-Based Affinity Chromatography Method for Antibody Purification via Nucleotide Binding Site Targeting
The conserved nucleotide binding site (NBS), found within
the Fab
variable domain of antibodies, remains a not-so-widely known and underutilized
site. Here we describe a novel affinity chromatography method that
utilizes the NBS as a target for selectively purifying antibodies
from complex mixtures. The affinity column was prepared by coupling
indole butyric acid (IBA), which has a monovalent affinity for the
NBS with a <i>K</i><sub>d</sub> ranging between 1 and 8
ÎŒM, to ToyoPearl resin resulting in the NBS targeting affinity
column (NBS<sup>IBA</sup>). The proof-of-concept studies performed
using the chimeric pharmaceutical antibody rituximab demonstrated
that antibodies were selectively captured and retained on the NBS<sup>IBA</sup> column and were successfully eluted by applying a mild
NaCl gradient at pH 7.0. Furthermore, the NBS<sup>IBA</sup> column
consistently yielded >95% antibody recovery with >98% purity,
even
when the antibody was purified from complex mixtures such as conditioned
cell culture supernatant, hybridoma media, and mouse ascites fluid.
The results presented in this study establish the NBS<sup>IBA</sup> column as a viable small-molecule-based affinity chromatography
method for antibody purification with significant implications in
industrial antibody production. Potential advantages of the NBS<sup>IBA</sup> platform are improved antibody batch quality, enhanced
column durability, and reduced overall production cost