Site-specific immobilization of an L-lactate dehydrogenase via an engineered surface cysteine residue

1993 ◽  
Vol 15 (1) ◽  
pp. 29-34 ◽  
Author(s):  
Helmut K. W. Kallwass ◽  
Wendy Parris ◽  
Emma L. A. Macfariane ◽  
Marvin Gold ◽  
J. Bryan Jones
Keyword(s):  
Lactate Dehydrogenase ◽  
Cysteine Residue ◽  
Site Specific

scholarly journals Analysis of relative bacterial activity and lactate dehydrogenase gene expression of caries-associated bacteria in a site-specific natural biofilm: an ex vivo study

2020 ◽  
Author(s):  
Carolin Walther ◽  
Sandra Zumbülte ◽  
Christoph M. Faerber ◽  
Richard Johannes Wierichs ◽  
Hendrik Meyer-Lueckel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactate Dehydrogenase ◽  
16S Rrna ◽  
Bacterial Activity ◽  
Oral Microbiome ◽  
Significant Activity ◽  
Site Specific ◽  
Associated Bacteria ◽  
16S Gene ◽  
A Site

Abstract Objectives Detecting bacterial activity is considered a promising approach to monitor shifts from symbiosis to dysbiosis in oral microbiome. The present study aimed at investigating both the relative bacterial activity and the lactate dehydrogenase (ldh) gene expression of caries-associated bacteria in a site-specific natural biofilm. Material and methods Sixty subjects (age, mean ± SE: 30.1 ± 1.4) were allocated to two groups: caries-free subjects (CF) or caries-active subjects (CA). CF presented one sound surface (CFS, n = 30). CA presented two donor sites: a cavitated caries lesion (CAC, n = 30) and a sound reference surface (CAS, n = 30). Real-time quantitative PCR (q-PCR) on species or genus level and total bacteria was performed targeting the 16S gene, the 16S rRNA, the ldh gene, and the ldh mRNA (increasing 16S ribosomal RNA copy numbers can function as an indicator of increased energy metabolism). As the 16S rRNA abundance represents the number of ribosomes, while the 16S gene abundance represents the number of genomes, the quotient of the relative abundances functions as a measure for the relative bacterial activity (%). Results Both lactobacilli and S. mutans showed the highest relative bacterial activity in CAC ((mean ± SE) 218 ± 60% and 61 ± 16%, respectively) and the lowest values for both sound reference surfaces (69 ± 48%; 8 ± 3%). Significant differences were found between CAC and CAS as well as between CAC and CFS for both lactobacilli and S. mutans (p < 0.05). The ldh gene expression of lactobacilli and S. mutans only showed moderate values in CAC (1.90E+03 ± 2.11E+03; 2.08E+04 ± 4.44E+04 transcripts/μl) and CFS (2.04E+03 ± 2.74E+03; 8.16E+03 ± 6.64E+03 transcripts/μl); consequently no significant differences were detected. Conclusion and clinical relevance Caries-associated bacteria (lactobacilli and S. mutans) showed the highest relative bacterial activity in plaque of cavitated lesions, the lowest in sound surfaces, allowing the detection of a significant activity shift in health and disease for caries-active patients. However, no significant differences in ldh gene expression could be determined.

scholarly journals REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants

2019 ◽  
Author(s):  
Kevin Dalton ◽  
Tom Lopez ◽  
Vijay Pande ◽  
Judith Frydman
Keyword(s):  
Rational Design ◽  
Biochemical Characterization ◽  
Cysteine Residue ◽  
Rational Approach ◽  
Protein Activity ◽  
Methanococcus Maripaludis ◽  
Site Specific ◽  
Protein Variants ◽  
Group Ii Chaperonin ◽  
Reduced Activity

AbstractSite-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.

Luminescent rhenium(I) perfluorobiphenyl complexes as site-specific labels for peptides to afford photofunctional bioconjugates

2021 ◽  
Author(s):  
Peter Kam-Keung Leung ◽  
Lawrence Cho-Cheung Lee ◽  
Tiffany Ka-Yan Ip ◽  
Hua-Wei Liu ◽  
Shek-Man Yiu ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Cysteine Residue ◽  
Site Specific

We report herein new luminescent rhenium(I) perfluorobiphenyl complexes that reacted specifically with the cysteine residue of the π-clamp sequence (FCPF) to afford novel peptide-based imaging reagents, photosensitisers for singlet oxygen...

Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)

Biomaterials ◽  
2013 ◽  
Vol 34 (36) ◽  
pp. 9115-9123 ◽  
Author(s):  
Li-Qiang Pan ◽  
Hai-Bin Wang ◽  
Jun Lai ◽  
Ying-Chun Xu ◽  
Chen Zhang ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Cysteine Residue ◽  
Site Specific ◽  
Necrosis Factor

scholarly journals Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

2009 ◽  
Vol 191 (6) ◽  
pp. 1933-1940 ◽  
Author(s):  
André Larouche ◽  
Paul H. Roy
Keyword(s):  
Antibiotic Resistance Genes ◽  
Cysteine Residue ◽  
Wild Type ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Environmental Bacterium ◽  
Target Sites ◽  
A Site

ABSTRACT Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.

Novel application of S-nitrosoglutathione–Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation

2000 ◽  
Vol 349 (2) ◽  
pp. 567-578
Author(s):  
Peter KLATT ◽  
Estela PINEDA MOLINA ◽  
Dolores PÉREZ-SALA ◽  
Santiago LAMAS
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Camp Response Element Binding ◽  
Cysteine Residue ◽  
Activator Protein 1 ◽  
Site Specific ◽  
Binding Domains ◽  
Nuclear Extracts ◽  
Protein Thiols

Site-specific S-glutathionylation is emerging as a novel mechanism by which S-nitrosoglutathione (GSNO) may modify functionally important protein thiols. Here, we show that GSNO-Sepharose mimicks site-specific S-glutathionylation of the transcription factors c-Jun and p50 by free GSNO in vitro. Both c-Jun and p50 were found to bind to immobilized GSNO through the formation of a mixed disulphide, involving a conserved cysteine residue located in the DNA-binding domains of these transcription factors. Furthermore, we show that c-Jun, p50, glycogen phosphorylase b, glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, glutaredoxin and caspase-3 can be precipitated from a mixture of purified thiol-containing proteins by the formation of a mixed-disulphide bond with GSNO-Sepharose. With few exceptions, protein binding to this matrix correlated well with the susceptibility of the investigated proteins to undergo GSNO- but not diamide-induced mixed-disulphide formation in vitro. Finally, it is shown that covalent GSNO-Sepharose chromatography of HeLa cell nuclear extracts results in the enrichment of proteins which incorporate glutathione in response to GSNO treatment. As suggested by DNA-binding assays, this group of nuclear proteins include the transcription factors activator protein-1, nuclear factor-ĸB and cAMP-response-element-binding protein. In conclusion, we introduce GSNO-Sepharose as a probe for site-specific S-glutathionylation and as a novel and potentially useful tool to isolate and identify proteins which are candidate targets for GSNO-induced mixed-disulphide formation.

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