Dataset Information

Recombinant expression and preliminary characterization of Peptidyl-prolyl cis/trans-isomerase Rrd1 from Saccharomyces cerevisiae.

ABSTRACT: Sacchromycescerevisiae Peptidyl-prolylcis/trans-isomerase Rrd1 has been linked to DNA repair, bud morphogenesis, advancement of the G1 phase, DNA replication stress, microtubule dynamics and is also necessary for the quick decrease in Sgs1p levels in response to rapamycin. In present study, Rrd1 gene was amplified by standard PCR and subsequently cloned downstream to bacteriophage T7 inducible promoter and lac operator of expression vector pET21d(+). Additionally, immobilized metal affinity chromatography (IMAC) was used to purify the protein upto its homogeneity, and its homogeneous purity was further confirmed through western blotting. Size exclusion chromatography implies that Rrd1 is existing as monomer in its natural state. Foldwise Rrd1 protein belongs to PTPA-like protein superfamily. Rrd1 showed characteristic negative minima at 222 and 208 nm represent protein typically acquired α helix in the far-UV CD spectra. Fluorescence spectra showed properly folded tertiary structures of Rrd1 at physiological conditions. Rrd1protein can be identified from different species using a fingerprint created by PIPSA analysis. The protein's abundance could aid in its crystallization, biophysical characterization and identification of other-interacting partners of Rrd1 protein.

SUBMITTER: Kashif M

PROVIDER: S-EPMC10263354 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Recombinant expression and preliminary characterization of Peptidyl-prolyl cis/trans-isomerase Rrd1 from Saccharomyces cerevisiae.

Kashif Mohd M Alsaiari Ahad Amer AA Kumar Bhupendra B Asalam Mohd M Khan Mohammad Imran MI Ahmad Abrar A Lone Rayees Ahmad RA Almehmadi Mazen M Zamzami Mazin A MA Akhtar Mohd Sohail MS

PloS one 20230613 6

Sacchromycescerevisiae Peptidyl-prolylcis/trans-isomerase Rrd1 has been linked to DNA repair, bud morphogenesis, advancement of the G1 phase, DNA replication stress, microtubule dynamics and is also necessary for the quick decrease in Sgs1p levels in response to rapamycin. In present study, Rrd1 gene was amplified by standard PCR and subsequently cloned downstream to bacteriophage T7 inducible promoter and lac operator of expression vector pET21d(+). Additionally, immobilized metal affinity chro ...[more]

PMID: 37310980

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Project description:BackgroundThe peptidyl-prolyl cis/trans isomerase (PPIase) class of proteins is present in all known eukaryotes, prokaryotes, and archaea, and it is comprised of three member families that share the ability to catalyze the cis/trans isomerisation of a prolyl bond. Some fungi have been used as model systems to investigate the role of PPIases within the cell, however how representative these repertoires are of other fungi or humans has not been fully investigated.ResultsPPIase numbers within these fungal repertoires appears associated with genome size and orthology between repertoires was found to be low. Phylogenetic analysis showed the single-domain FKBPs to evolve prior to the multi-domain FKBPs, whereas the multi-domain cyclophilins appear to evolve throughout cyclophilin evolution. A comparison of their known functions has identified, besides a common role within protein folding, multiple roles for the cyclophilins within pre-mRNA splicing and cellular signalling, and within transcription and cell cycle regulation for the parvulins. However, no such commonality was found with the FKBPs. Twelve of the 17 human cyclophilins and both human parvulins, but only one of the 13 human FKBPs, identified orthologues within these fungi. hPar14 orthologues were restricted to the Pezizomycotina fungi, and R. oryzae is unique in the known fungi in possessing an hCyp33 orthologue and a TPR-containing FKBP. The repertoires of Cryptococcus neoformans, Aspergillus fumigatus, and Aspergillus nidulans were found to exhibit the highest orthology to the human repertoire, and Saccharomyces cerevisiae one of the lowest.ConclusionGiven this data, we would hypothesize that: (i) the evolution of the fungal PPIases is driven, at least in part, by the size of the proteome, (ii) evolutionary pressures differ both between the different PPIase families and the different fungi, and (iii) whilst the cyclophilins and parvulins have evolved to perform conserved functions, the FKBPs have evolved to perform more variable roles. Also, the repertoire of Cryptococcus neoformans may represent a better model fungal system within which to study the functions of the PPIases as its genome size and genetic tractability are equal to those of Saccharomyces cerevisiae, whilst its repertoires exhibits greater orthology to that of humans. However, further experimental investigations are required to confirm this.

Dataset Information

Recombinant expression and preliminary characterization of Peptidyl-prolyl cis/trans-isomerase Rrd1 from Saccharomyces cerevisiae.

Publications

Recombinant expression and preliminary characterization of Peptidyl-prolyl cis/trans-isomerase Rrd1 from Saccharomyces cerevisiae.

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