Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

Novel Heterozygous Mutations in ZP2 Cause Abnormal Zona Pellucida and Female Infertility.

Zona pellucida (ZP) which is an extracellular matrix consisting of ZP1, ZP2, ZP3, and ZP4 plays a vital role in oocyte maturity, early embryonic development, and fertilization process. Any alterations of structure or function may lead to the abnormal formation of ZP and female infertility. Two novel heterozygous mutations c.1859G > A (p.Cys620Tyr) and c.1421 T > C (p.Leu474Pro) in ZP2 gene were recognized in three patients from two unrelated families with abnormal ZP and female infertility in this study. The expression constructs carrying wild-type ZP2 gene, c.1859G > A (p.Cys620Tyr) mutant ZP2 gene, and c.1421 T > C (p.Leu474Pro) mutant ZP2 gene were transfected into CHO cells respectively. There was a remarkable decrease in the expression of p.Cys620Tyr mutant protein with western blot. In addition, secretion of p.Leu474Pro mutant protein in the culture medium reduced markedly compared with that of wild-type ZP2 protein. Furthermore, co-immunoprecipitation showed that the p.Leu474Pro mutation affected the interaction between ZP2 and ZP3. Prediction of three-dimensional (3D) structure of the proteins showed that p.Cys620Tyr mutation altered the disulfide bond of ZP2 protein and may affect its function. These findings extend the ranges of mutations of ZP2 gene. Meanwhile, it will be helpful to the precise diagnosis of abnormal ZP.

Clinical significance of immunohistochemistry to detect BRAF V600E mutant protein in thyroid tissues.

ABSTRACT: This study investigated the feasibility of using immunohistochemistry (IHC) instead of PCR to detect BRAF V600E mutant protein in papillary thyroid carcinoma (PTC), and to determine the value of using preoperative BRAF V600E mutant protein by IHC to assist in the diagnosis of thyroid nodule patients with Hashimoto's thyroiditis (HT).The expression of BRAFV600E mutant protein was measured in 23 cases of HT+PTC, 31 cases of PTC, and 28 cases of HT by IHC, followed by PCR in the same samples for validation. SPSS 19.0 software was used for statistical analysis.The sensitivity and specificity of IHC to detect BRAF V600E mutation were 100% and 42.86%, respectively. In addition, the mutation rate of BRAF V600E protein in the HT+PTC group (34.78%, 8/23) was lower than that in the PTC group (80.65%, 25/31).The application of IHC to detect BRAF V600E mutant protein has good sensitivity but not specificity to diagnose PTC. IHC can be used as a preliminary screening method to detect BRAF V600E mutation. The strongly positive (+++) staining of IHC potently indicated BRAF V600E gene mutation. For suspicious thyroid nodules combined with HT, the detection of BRAF V600E mutant protein with IHC alone is not of great significance for differentiating benign and malignant nodules.

Substrate structure and computation guided engineering of a lipase for omega-3 fatty acid selectivity.

Enrichment of omega-3 fatty acids (É·-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky É·-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the É·-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity.

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production.

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Proteome-wide onco-proteogenomic somatic variant identification in ER-positive breast cancer.

BACKGROUND: Recent advances in mass spectrometric instrumentation and bioinformatics have critically contributed to the field of proteogenomics. Nonetheless, whether that integrative approach has reached the point of maturity to effectively reveal the flow of genetic variants from DNA to proteins still remains elusive. The objective of this study was to detect somatically acquired protein variants in breast cancer specimens for which full genome and transcriptome data was already available (BASIS cohort). METHODS: LC-MS/MS shotgun proteomic results of 21 breast cancer tissues were coupled to DNA sequencing data to identify variants at the protein level and finally were used to associate protein expression with gene expression levels. RESULTS: Here we report the observation of three sequencing-predicted single amino acid somatic variants. The sensitivity of single amino acid variant (SAAV) detection based on DNA sequencing-predicted single nucleotide variants was 0.4%. This sensitivity was increased to 0.6% when all the predicted variants were filtered for MS "compatibility" and was further increased to 2.9% when only proteins with at least one wild type peptide detected were taken into account. A correlation of mRNA abundance and variant peptide detection revealed that transcripts for which variant proteins were detected ranked among the top 6.3% most abundant transcripts. The variants were detected in highly abundant proteins as well, thus establishing transcript and protein abundance and MS "compatibility" as the main factors affecting variant onco-proteogenomic identification. CONCLUSIONS: While proteomics fails to identify the vast majority of exome DNA variants in the resulting proteome, its ability to detect a small subset of SAAVs could prove valuable for precision medicine applications.

Surface enhanced Raman spectroscopy based immunosensor for ultrasensitive and selective detection of wild type p53 and mutant p53R175H.

p53 is a powerful transcription factor playing a pivotal role in the prevention of cancer development and in maintaining genome integrity. This oncosuppressor is found to be functionally inactivated by mutations in many human tumors. Accordingly, wild type p53 and its oncogenic mutants represent valuable cancer biomarkers for diagnostic and prognostic purposes. We developed a highly sensitive biosensor, based on Surface Enhanced Raman Spectroscopy, for detection of wild type p53 and of p53R175H, which is one of the most frequent tumor-associated mutants of p53. Our approach combines the huge Raman signal enhancement, mainly arising from the plasmonic resonance effect on molecules close to gold nanoparticles, with the antigen-antibody biorecognition specificity. By following the enhanced signal of a specific Raman marker, intrinsic to the nanoparticle-antibody bioconjugation, we were able to push the antigen detection level down to the attomolar range in buffer and to the femtomolar range in spiked human serum. The method demonstrated a high reproducibility and a remarkable selectivity in discriminating between wild type p53 and p53R175H mutant, in both buffer and serum. A calibration plot was built and validated by ELISA for a reliable quantification of p53. These findings entitle our SERS-based immunosensor as a powerful and reliable tool for a non-invasive screening in human serum targeting p53 network. The approach could be easily extended to ultrasensitive detection of other markers of general interest, with feasible implementations into multiplex assays, functioning as lab-on-chip devices for several applications.

Increased 4R tau expression and behavioural changes in a novel MAPT-N296H genomic mouse model of tauopathy.

The microtubule-associated protein tau is implicated in various neurodegenerative diseases including Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, which are characterized by intracellular accumulation of hyperphosphorylated tau. Mutations in the tau gene MAPT cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In the human central nervous system, six tau isoforms are expressed, and imbalances in tau isoform ratios are associated with pathology. To date, few animal models of tauopathy allow for the potential influence of these protein isoforms, relying instead on cDNA-based transgene expression. Using the P1-derived artificial chromosome (PAC) technology, we created mouse lines expressing all six tau isoforms from the human MAPT locus, harbouring either the wild-type sequence or the disease-associated N296H mutation on an endogenous Mapt-/- background. Animals expressing N296H mutant tau recapitulated early key features of tauopathic disease, including a tau isoform imbalance and tau hyperphosphorylation in the absence of somatodendritic tau inclusions. Furthermore, N296H animals displayed behavioural anomalies such as hyperactivity, increased time in the open arms of the elevated plus maze and increased immobility during the tail suspension test. The mouse models described provide an excellent model to study the function of wild-type or mutant tau in a highly physiological setting.

Expression and function of NKp46 W32R: the human homologous protein of mouse NKp46 W32R (Noé).

Natural killer (NK) cells eradicate infected cells and tumors following the triggering of activating receptors, like the Natural Cytotoxicity Receptors (NCRs), which include NKp30, NKp44 and NKp46. NKp46 is the only NCR expressed in mice (mNKp46), and except for some Innate Lymphoid Cell (ILC) populations (ILC1/3 subsets), its expression is restricted to NK cells. Previously, a mouse named Noé was generated in which a random point mutation (W32R) impaired the cell surface expression of mNKp46. Interestingly, the Noé mice NK cells expressed twice as much of the transcription factor Helios, and displayed general non-NKp46 specific hyperactivity. We recently showed that the mNKp46 W32R (Noé) protein was expressed on the surface of various cells; albeit slowly and unstably, that it is aberrantly glycosylated and accumulates in the ER. Interestingly, the Tryptophan (Trp) residue in position 32 is conserved between humans and mice. Therefore, we studied here the human orthologue protein of mNKp46 W32R, the human NKp46 W32R. We demonstrated that NKp46 W32R is aberrantly glycosylated, accumulates in the ER, and is unstable on the cell surface. Furthermore, we showed that overexpression of NKp46 W32R or Helios resulted in augmented NK cell activation, which may be applied to boost NK activity for therapeutic applications.

Sequential treatment with immunotherapy and BRAF inhibitors in BRAF-mutant advanced melanoma

Purpose. Immunotherapy (IT) agents and BRAF inhibitors (BRAFi) are effective treatments for patients with advanced BRAF-mutant melanoma although the optimal sequence remains to be elucidated. The aim of this study was to compare the outcomes of two different cohorts of patients treated with BRAFi first, then IT or the reverse sequence. Patients and methods. This is a retrospective study on two groups of patients: a cohort was treated first with BRAFi followed by immunotherapy (BRAFi-IT) and the other cohort with the reverse sequence (IT-BRAFi). Baseline characteristics and clinical outcomes were compared between the two cohorts. Results. A total of 25 patients were included in the study. Sixteen patients were given BRAFi-IT sequence and nine received IT-BRAFi sequence. No differences were observed in the characteristics of patients prior to each treatment between cohorts. Objective response rate (ORR) achieved by BRAFi were not different among groups. ORR achieved by IT was higher when administered after BRAFi (43.8 vs 0 %). Survival rates at 1-2 years were similar in both cohorts and median overall survival was not different for BRAFi-IT and IT-BRAFi (log rank test p = 0.97). Conclusions. No differences were observed in OS between the two cohorts. These results support the indistinct use of IT or BRAFi as initial treatment in patients with metastatic BRAF-mutant melanoma, although higher rate of response to IT was observed when administered after BRAFi. Prospective randomized clinical trials are needed on this issue (AU)

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Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies.

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.

Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface.

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.

Mutant Proteogenomics.

Identification of mutant proteins in biological samples is one of the emerging areas of proteogenomics. Despite the fact that only a limited number of studies have been published up to now, it has the potential to recognize novel disease biomarkers that have unique structure and desirably high specificity. Such properties would identify mutant proteoforms related to diseases as optimal drug targets useful for future therapeutic strategies. While mass spectrometry has demonstrated its outstanding analytical power in proteomics, the most frequently applied bottom-up strategy is not suitable for the detection of mutant proteins if only databases with consensus sequences are searched. It is likely that many unassigned tandem mass spectra of tryptic peptides originate from single amino acid variants (SAAVs). To address this problem, a couple of protein databases have been constructed that include canonical and SAAV sequences, allowing for the observation of mutant proteoforms in mass spectral data for the first time. Since the resulting large search space may compromise the probability of identifications, a novel concept was proposed that included identification as well as verification strategies. Together with transcriptome based approaches, targeted proteomics appears to be a suitable method for the verification of initial identifications in databases and can also provide quantitative insights to expression profiles, which often reflect disease progression. Important applications in the field of mutant proteoform identification have already highlighted novel biomarkers in large-scale investigations.

Real-time imaging of Huntingtin aggregates diverting target search and gene transcription.

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.

Aspergillus fumigatus spore proteomics and genetics reveal that VeA represses DefA-mediated DNA damage response.

Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues.

Detection of amino acid substitutions in the GyrA protein of fluoroquinolone-resistant typhoidal Salmonella isolates using high-resolution mass spectrometry.

Infections with typhoidal Salmonella isolates that are resistant to fluoroquinolone antibiotics have become very common in several Asian countries. In the majority of these cases, resistance to fluoroquinolone-based antibiotics is associated with genetic mutations in the quinolone resistance-determining region (QRDR) of the bacterial DNA gyrase gene gyrA. The objective of this study was to detect these amino acid substitutions by high-resolution mass spectrometry instead of sequencing of the gyrA gene. A liquid chromatography-mass spectrometry (LC-MS) methodology was developed and evaluated for the detection of amino acid substitutions in the GyrA protein of 23 typhoidal Salmonella isolates. These isolates included typhoidal Salmonella that possessed different antibiotic sensitivities to fluoroquinolone antibiotics. The LC-MS methodology correctly identified peptide sequences associated with phenotypic QRDR mutations of the GyrA protein in all 23 phenotypically diverse typhoidal Salmonella isolates tested. In conclusion, a reliable and rapid LC-MS methodology has been developed that is able to identify GyrA QRDR mutations that are involved in the development of fluoroquinolone resistance in typhoidal Salmonella spp. Furthermore, this 'proof of principle' study indicates the potential usefulness of LC-MS in future detection of antibiotic resistance.

Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq.

Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio - OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this 'internal standard' calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.

A selection assay for haloalkane dehalogenase activity based on toxic substrates.

Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 10(6) cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases.