A machine-learning-guided mutagenesis platform for accelerated discovery of novel functional proteins

AbstractMolecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space that requires high costs for screening experiments. Here, we propose a novel approach that combines molecular evolution with machine learning. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed to yellow while improving its fluorescence intensity. Using 155 and 78 variants for the initial and the second-round libraries, respectively, we successfully obtained a number of proteins showing yellow fluorescence, 12 of which had better fluorescence performance than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful platform for accelerated discovery of functional proteins.

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Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants

Journal of Cell Science ◽

10.1242/jcs.115.6.1221 ◽

2002 ◽

Vol 115 (6) ◽

pp. 1221-1229 ◽

Cited By ~ 6

Author(s):

Tomoo Ohashi ◽

Daniel P. Kiehart ◽

Harold P. Erickson

Keyword(s):

Actin Cytoskeleton ◽

Actin Filament ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Actin Binding ◽

Matrix Assembly ◽

Actin Binding Domain ◽

Filament Bundles ◽

Green Fluorescent ◽

Protein Variants

We have prepared 3T3 cells doubly labeled to visualize simultaneously the extracellular fibronectin (FN) matrix and intracellular actin cytoskeleton in living cell cultures. We used FN-yellow fluorescent protein (FN-yfp) for the FN matrix, and the actin-binding domain of moesin fused to cyan fluorescent protein (cfp-Moe) to stain actin. Actin filament bundles were clearly seen in the protruding lamellae of the cells. FN matrix assembly appeared to be initiated as small spots of FN at the ends of actin filament bundles. The spots then elongated along the actin filament bundle toward the cell center to form FN fibrils. The end of the fibril towards the cell edge appeared immobile, and probably attached to the substrate, whereas the end toward the cell center frequently showed movements, suggesting attachment to the cell. Combining our data with the observations of Pankov et al. we suggest that fibrils grow by stretching this mobile end toward the cell center while adding new FN molecules at the end and along the entire lenght. When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched.

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Halide and Proton Binding Kinetics of Yellow Fluorescent Protein Variants

Biochemistry ◽

10.1021/bi3016839 ◽

2013 ◽

Vol 52 (14) ◽

pp. 2482-2491 ◽

Cited By ~ 3

Author(s):

Harriet E. Seward ◽

Jaswir Basran ◽

Roanne Denton ◽

Mark Pfuhl ◽

Frederick W. Muskett ◽

...

Keyword(s):

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Binding Kinetics ◽

Proton Binding ◽

Protein Variants ◽

Kinetics Of

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A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

Biochemical Journal ◽

10.1042/bj20111300 ◽

2011 ◽

Vol 441 (1) ◽

pp. 209-217 ◽

Cited By ~ 27

Author(s):

Iraia García-Santisteban ◽

Sonia Bañuelos ◽

Jose A. Rodríguez

Keyword(s):

Nuclear Export ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Site Directed Mutagenesis ◽

The Novel ◽

Sequence Motifs ◽

Leptomycin B ◽

Specific Amino Acid ◽

Green Fluorescent ◽

Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

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A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

Journal of Food Protection ◽

10.4315/0362-028x-72.7.1513 ◽

2009 ◽

Vol 72 (7) ◽

pp. 1513-1520 ◽

Cited By ~ 63

Author(s):

MANAN SHARMA ◽

DAVID T. INGRAM ◽

JITENDRA R. PATEL ◽

PATRICIA D. MILLNER ◽

XIAOLIN WANG ◽

...

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Plasmid Vector ◽

Green Fluorescent Protein Gene ◽

Escherichia Coli O157 ◽

E Coli ◽

Novel Approach ◽

Efficient Uptake ◽

Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

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High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00884.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. H1647-H1654 ◽

Cited By ~ 11

Author(s):

Jean-Philippe Fortin ◽

Johanne Bouthillier ◽

François Marceau

Keyword(s):

Fluorescent Protein ◽

Cultured Cells ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Brefeldin A ◽

Metabolic Inhibitors ◽

Maximal Effect ◽

Hek 293 ◽

B1 Receptors ◽

Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

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Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons

Molecular Biology of the Cell ◽

10.1091/mbc.11.4.1213 ◽

2000 ◽

Vol 11 (4) ◽

pp. 1213-1224 ◽

Cited By ~ 180

Author(s):

Christoph Kaether ◽

Paul Skehel ◽

Carlos G. Dotti

Keyword(s):

Membrane Proteins ◽

Hippocampal Neurons ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Microscopy Study ◽

Video Microscopy ◽

Axonal Membrane ◽

Color Video ◽

Green Fluorescent ◽

Living Neurons

Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 μm/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 μm/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier.

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pH of the Cytoplasm and Periplasm of Escherichia coli: Rapid Measurement by Green Fluorescent Protein Fluorimetry

Journal of Bacteriology ◽

10.1128/jb.00615-07 ◽

2007 ◽

Vol 189 (15) ◽

pp. 5601-5607 ◽

Cited By ~ 199

Author(s):

Jessica C. Wilks ◽

Joan L. Slonczewski

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Time Resolution ◽

Fluorescent Protein ◽

Osmotic Shock ◽

Yellow Fluorescent Protein ◽

Fluorescence Signal ◽

Cytoplasmic Ph ◽

Green Fluorescent ◽

External Ph

ABSTRACT Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.

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Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

The Journal of Cell Biology ◽

10.1083/jcb.200109030 ◽

2002 ◽

Vol 156 (3) ◽

pp. 511-518 ◽

Cited By ~ 211

Author(s):

Pierre Barbero ◽

Lenka Bittova ◽

Suzanne R. Pfeffer

Keyword(s):

Fluorescent Protein ◽

Cultured Cells ◽

Late Endosome ◽

Live Cells ◽

Transport Vesicles ◽

Late Endosomes ◽

Mannose 6 Phosphate ◽

Green Fluorescent ◽

Golgi Network ◽

Protein Variants

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

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Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

Applied and Environmental Microbiology ◽

10.1128/aem.00136-13 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2218-2224 ◽

Cited By ~ 105

Author(s):

Jeffrey L. Bose ◽

Paul D. Fey ◽

Kenneth W. Bayles

Keyword(s):

Staphylococcus Aureus ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Exchange System ◽

Fluorescent Reporter ◽

Content Type ◽

Genetic Tools ◽

Allelic Exchange ◽

Green Fluorescent

ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

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