A Novel Actinobacterial Cutinase Containing a Non-Catalytic Polymer-Binding Domain

2021 ◽  
Author(s):  
Kofi Abokitse ◽  
Stephan Grosse ◽  
Hannes Leisch ◽  
Christopher R. Corbeil ◽  
Florence Perrin-Sarazin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Textile Industry ◽  
Thermophilic Bacteria ◽  
Genome Mining ◽  
Pectate Lyase ◽  
Phytopathogenic Fungi ◽  
Binding Domain ◽  
Amino Acid Region ◽  
Hydroxyl Fatty Acids

The single putative cutinase-encoding gene from the genome of Kineococcus radiotolerans SRS30216 was cloned and expressed in Escherichia coli as a secreted fusion protein, designated YebF-KrCUT, where YebF is the extracellular carrier protein. The 294-amino acid sequence of KrCUT is unique among currently characterized cutinases by having a C-terminal extension that consists of a short (Pro-Thr)-rich linker and a 55-amino-acid region resembling the substrate binding domain of poly(hydroxybutyrate) (PHB) depolymerases. Phylogenetically, KrCUT takes a unique position among known cutinases and cutinase-like proteins of bacterial and fungal origin. A modeled structure of KrCUT, although displaying a typical α/ß hydrolase fold, shows some unique loops close to the catalytic site. The 39-kDa YebF-KrCUT fusion protein and a truncated variant thereof were purified to electrophoretic homogeneity and functionally characterized. The melting temperatures ( T m ) of KrCUT and its variant KrCUT206 devoid of the putative PHB-binding domain were established to be very similar at 50–51°C. Cutinase activity was confirmed by the appearance of characteristic cutin components, C 16 and C 18 hydroxyl fatty acids, in the mass chromatograms following incubation of KrCUT with apple cutin as substrate. KrCUT also efficiently degraded synthetic polyesters such as polycaprolactone and poly(1,3-propylene adipate). Although incapable of PHB depolymerization, KrCUT could efficiently bind PHB, confirming the predicted characteristic of the C-terminal region. KrCUT also potentiated the activity of pectate lyase in the degradation of pectin from hemp fibres. This synergistic effect is relevant to the enzyme retting process of natural fibres. IMPORTANCE. To date only a limited number of cutinases have been isolated and characterized from nature, the majority being sourced from phytopathogenic fungi and thermophilic bacteria. The significance of our research relates to the identification and characterization of a unique member of microbial cutinases, of name KrCUT, that was derived from the genome of the Gram-positive Kineococcus radiotolerans SRS30216, a highly radiation-resistant actinobacterium. Given the wide-ranging importance of cutinases in applications such as the degradation of natural and synthetic polymers, in the textile industry, in laundry detergents, or in biocatalysis (e.g., transesterification reactions), our results could foster new research leading to broader biotechnological impacts. This study also demonstrated that genome mining or prospecting is a viable means to discover novel biocatalysts as environmentally friendly and biotechnological tool.

Construction and immunogenic studies of a mFc fusion receptor binding domain (RBD) of spike protein as a subunit vaccine against SARS-CoV-2 infection

2020 ◽  
Vol 56 (61) ◽  
pp. 8683-8686 ◽  
Author(s):  
Xiaoxiao Qi ◽  
Bixia Ke ◽  
Qian Feng ◽  
Deying Yang ◽  
Qinghai Lian ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Subunit Vaccine ◽  
Binding Domain ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Humoral And Cellular Immunity ◽  
A Subunit

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice.

scholarly journals A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors

2003 ◽  
Vol 30 (2) ◽  
pp. 197-211 ◽  
Author(s):  
S Chopin-Delannoy ◽  
S Thenot ◽  
F Delaunay ◽  
E Buisine ◽  
A Begue ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Nuclear Receptors ◽  
Nuclear Localization ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Fusion ◽  
Orphan Receptors ◽  
Amino Terminal

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

scholarly journals Functional and conserved domains of the Drosophila transcription factor encoded by the segmentation gene knirps

1994 ◽  
Vol 14 (12) ◽  
pp. 7899-7908
Author(s):  
N Gerwin ◽  
A La Rosée ◽  
F Sauer ◽  
H P Halbritter ◽  
M Neumann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Broad Band ◽  
Binding Domain ◽  
Orphan Receptor ◽  
Dna Binding Domain ◽  
Coding Region ◽  
Amino Acid Region

The Drosophila gap gene knirps (kni) is required for abdominal segmentation. It encodes a steroid/thyroid orphan receptor-type transcription factor which is distributed in a broad band of nuclei in the posterior region of the blastoderm. To identify essential domains of the kni protein (KNI), we cloned and sequenced the DNA encompassing the coding region of nine kni mutant alleles of different strength and kni-homologous genes of related insect species. We also examined in vitro-modified versions of KNI in various assay systems both in vitro and in tissue culture. The results show that KNI contains several functional domains which are arranged in a modular fashion. The N-terminal 185-amino-acid region which includes the DNA-binding domain and a functional nuclear location signal fails to provide kni activity to the embryo. However, a truncated KNI protein that contains additional 47 amino acids exerts rather strong kni activity which is functionally defined by a weak kni mutant phenotype of the embryo. The additional 47-amino-acid stretch includes a transcriptional repressor domain which acts in the context of a heterologous DNA-binding domain of the yeast transcriptional activator GAL4. The different domains of KNI as defined by functional studies are conserved during insect evolution.

Non-neuronal 210 × 10(3) Mr microtubule-associated protein (MAP4) contains a domain homologous to the microtubule-binding domains of neuronal MAP2 and tau

1991 ◽  
Vol 98 (1) ◽  
pp. 27-36
Author(s):  
S.J. Chapin ◽  
J.C. Bulinski
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Hela Cell ◽  
Fusion Proteins ◽  
Fetal Brain ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Projection Domain

A polyclonal antiserum raised against a HeLa cell microtubule-associated protein of Mr 210,000 (210 kD MAP or MAP4), an abundant non-neuronal MAP, was used to isolate cDNA clones encoding MAP4 from a human fetal brain lambda gt11 cDNA expression library. The largest of these clones, pMAP4.245, contains an insert of 4.1 kb and encodes a 245 kD beta-galactosidase fusion protein. Evidence that pMAP4.245 encodes MAP4 sequences includes immunoabsorption of MAP4 antibodies with the pMAP4.245 fusion protein, as well as identity of protein sequences obtained from HeLa 210 kD MAP4 with amino acid sequences encoded by pMAP4.245. The MAP4.245 cDNA hybridizes to several large (approximately 6–9 kb) transcripts on Northern blots of HeLa cell RNA. DNA sequencing of overlapping MAP4 cDNA clones revealed a long open reading frame containing a C-terminal region with three imperfect 18-amino acid repeats; this region is homologous to a motif present in the microtubule (MT)-binding domain of two prominent neuronal MAPs, MAP2 and tau. The pMAP4.245 sequence also encoded a series of unrelated repeats, located in the MAP's projection domain, N-terminal to the MT-binding domain. MAP4.245 fusion proteins bound to MTs in vitro, while fusion proteins that contained only the projection domain repeats failed to bind specifically to MTs. Thus, the major human non-neuronal MAP resembles two neuronal MAPs in its MT-binding domain, while most of the molecule has sequences, and presumably functions, distinct from those of the neuronal MAPs.

scholarly journals Domain Interactions between Coregulator ARA70 and the Androgen Receptor (AR)

2002 ◽  
Vol 16 (2) ◽  
pp. 287-300 ◽  
Author(s):  
Zhong-xun Zhou ◽  
Bin He ◽  
Susan H. Hall ◽  
Elizabeth M. Wilson ◽  
Frank S. French
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Luciferase Reporter ◽  
Affinity Matrix ◽  
Glutathione S Transferase ◽  
Transcriptional Activation Domain ◽  
Amino Acid Region ◽  
Tumor Virus

Abstract The coregulator function of AR-associated protein 70 (ARA70) was investigated to further characterize its interaction with the AR. Using a yeast two-hybrid assay, androgen-dependent binding of ARA70 deletion mutants to the AR ligand-binding domain (LBD) was strongest with ARA70 amino acids 321–441 of the 614 amino acid ARA70 protein. Mutations adjacent to or within an FxxLF motif in this 120-amino acid region abolished androgen-dependent binding to the AR-LBD both in yeast and in glutathione-S-transferase affinity matrix assays. Yeast one-hybrid assays revealed an intrinsic ARA70 transcriptional activation domain within amino acids 296–441. In yeast assays the ARA70 domains for transcriptional activation and for binding to the AR-LBD were inhibited by the C-terminal region of ARA70. Full-length ARA70 increased androgen-dependent AR transactivation in transient cotransfection assays using a mouse mammary tumor virus-luciferase reporter in CV1 cells. ARA70 also increased constitutive transcriptional activity of an AR NH2-terminal-DNA binding domain fragment and bound this region in glutathione-S-transferase affinity matrix assays. Binding was independent of the ARA70 FxxLF motif. The results identify an ARA70 motif required for androgen-dependent interaction with the AR-LBD and demonstrate that ARA70 can interact with the NH2-terminal and carboxyl-terminal regions of AR.

scholarly journals Functional and conserved domains of the Drosophila transcription factor encoded by the segmentation gene knirps.

1994 ◽  
Vol 14 (12) ◽  
pp. 7899-7908 ◽  
Author(s):  
N Gerwin ◽  
A La Rosée ◽  
F Sauer ◽  
H P Halbritter ◽  
M Neumann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Broad Band ◽  
Binding Domain ◽  
Orphan Receptor ◽  
Dna Binding Domain ◽  
Coding Region ◽  
Amino Acid Region

The Drosophila gap gene knirps (kni) is required for abdominal segmentation. It encodes a steroid/thyroid orphan receptor-type transcription factor which is distributed in a broad band of nuclei in the posterior region of the blastoderm. To identify essential domains of the kni protein (KNI), we cloned and sequenced the DNA encompassing the coding region of nine kni mutant alleles of different strength and kni-homologous genes of related insect species. We also examined in vitro-modified versions of KNI in various assay systems both in vitro and in tissue culture. The results show that KNI contains several functional domains which are arranged in a modular fashion. The N-terminal 185-amino-acid region which includes the DNA-binding domain and a functional nuclear location signal fails to provide kni activity to the embryo. However, a truncated KNI protein that contains additional 47 amino acids exerts rather strong kni activity which is functionally defined by a weak kni mutant phenotype of the embryo. The additional 47-amino-acid stretch includes a transcriptional repressor domain which acts in the context of a heterologous DNA-binding domain of the yeast transcriptional activator GAL4. The different domains of KNI as defined by functional studies are conserved during insect evolution.

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