Characterization of non-specific protein adsorption induced by triazole groups on the chromatography media using Cu (I)-catalyzed alkyne-azide cycloaddition reaction for ligand immobilization

ABSTRACT AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81 His-1Ile ) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

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Characterization of an activated human ros gene.

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3109 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3109-3116 ◽

Cited By ~ 61

Author(s):

C Birchmeier ◽

D Birnbaum ◽

G Waitches ◽

O Fasano ◽

M Wigler

Keyword(s):

Gene Transfer ◽

Protein Kinases ◽

Transmembrane Domain ◽

Extracellular Domain ◽

Kinase Domain ◽

Specific Protein ◽

Normal Human

A human oncogene, mcf3, previously detected by a combination of DNA-mediated gene transfer and a tumorigenicity assay, derives from a human homology of the avian v-ros oncogene. Both v-ros and mcf3 can encode a protein with homology to tyrosine-specific protein kinases, and both mcf3 and v-ros encode a potential transmembrane domain N terminal to the kinase domain. mcf3 probably arose during gene transfer from a normal human ros gene by the loss of a putative extracellular domain. There do not appear to be any other gross rearrangements in the structure of mcf3.

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Synthesis and Characterization of Multifunctional Interpenetrating Polymer and Its Applications in Protein Adsorption and Magnetite Loading

ChemistrySelect ◽

10.1002/slct.201700878 ◽

2017 ◽

Vol 2 (21) ◽

pp. 5895-5904 ◽

Cited By ~ 4

Author(s):

Xiaoxiao Li ◽

Xueting Li ◽

Xihua Lu

Keyword(s):

Protein Adsorption ◽

Synthesis And Characterization

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A Novel Chromodomain Protein, Pdd3p, Associates with Internal Eliminated Sequences during Macronuclear Development inTetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.20.11.4128-4134.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 4128-4134 ◽

Cited By ~ 47

Author(s):

Mikhail A. Nikiforov ◽

Martin A. Gorovsky ◽

C. David Allis

Keyword(s):

De Novo ◽

Germ Line ◽

Chromosome Breakage ◽

Specific Protein ◽

Developmentally Regulated ◽

Dna Elimination ◽

Internal Eliminated Sequences ◽

Subsequent Elimination ◽

Rearrangement Processes

ABSTRACT Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

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