Biosynthesis of human β2-adrenergic receptor in methylotrophic yeast Pichia pastoris and its purification

Gerasimov, Andrey; Zeinalov, O. A.; Eldarov, M. A.; Shulga, A. A.

doi:10.1134/s0026893312020057

Cited by 3 publications

(3 citation statements)

References 34 publications

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“…This choice of primer design was based on an assumption that hypothetical S. altissima SLAH3 gene should be also closely related to the two genus Suaeda ortholog genes SfSLAH3 and SgSLAH3, which are highly identical between themselves. These primers contained 5′-overhang tails for Gibson assembly cloning into pVR2ADRHis vector (Gerasimov et. al.…”

Section: Resultsmentioning

confidence: 99%

Molecular Cloning of Saslah3, a Novel Slow Anion Channel Slac/slah Family Gene, From Euhalophyte Suaeda Altissima (L.) Pall and Analysis of the Encoded Protein

Rostovtseva,

Khramov,

Myasoedov

et al. 2024

Preprint

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Cloning of the SaSLAH3 gene (OQ271227.1), an AtSLAH3 ortholog from the slow anion channels family (SLAC/SLAH), that was originally identified in Arabidopsis thaliana, has been carried out from the euhalophyte Suaeda altissima. Under hydroponic conditions, this species related to Amaranthaceae/Chenopodiaceae is capable to complete life cycle on nutrient solutions containing NaCl at high concentrations up to 1 M. Cloning of SaSLAH3 was performed based on a putative similarity of SaSLAH3 with the SLAH3 homologs from the related species Suaeda glauca and Suaeda fruticosa, using transcriptomes assembled previously. Membrane topology, analyzed by in silico approach, the 3-D structure, presence of conservative phenylalanine-bearing motives and as well as MEME-generated sequence motif patterns of SaSLAH3, all of these features were found typical for the SLAC/SLAH channels and had validated SaSLAH3 belonging to this family. Analysis of SLAC/SLAH phylogenetic relationship demonstrated the evolutionary divergence of SLAC-like proteins into three distinct groups characteristic of this family with location of SaSLAH3 in SLAH2/3 clade. Investigation of the expression profile by quantitative Real-Time PCR revealed increasing SaSLAH3 expression in roots and leaves upon elevation of NaCl concentration in nutrient solution under conditions of both sufficient nitrate supply and nitrate deficiency. SaSLAH3 is suggested to play an important role in enhancing chloride loading into root xylem and, thereafter in chloride delivery to shoot under salinity. Another physiological role of SaSLAH3 may be attributed to regulation of nitrate concentration in cytoplasm to ensure proper nitrogen nutrition of S. altissima plants under conditions of stiff competition of nitrate with chloride.

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Section: Resultsmentioning

confidence: 99%

Molecular Cloning of Saslah3, a Novel Slow Anion Channel Slac/slah Family Gene, From Euhalophyte Suaeda Altissima (L.) Pall and Analysis of the Encoded Protein

Rostovtseva,

Khramov,

Myasoedov

et al. 2024

Preprint

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“…Full-length SaCLCa2 and SaCLCc2 cDNAs were amplified with a CloneAmpPCR PreMix kit (Clontech, Mountain View, CA, USA) using the pairs of primers: pVR2_SaCLCa2_F and pVR2_SaCLCa2_R, pVR2_SaCLCc2_F and pVR2_SaCLCc2_R ( Table S3 ), and total first strand cDNA as a template. The amplified SaCLCa2 and SaCLCc2 cDNAs fragments were cloned into yeast vector pVR2 [ 59 ] using Gibson Assembly Cloning Kit (NEB, USA), and sequenced. For expression in the yeast mutant Δgef1 , the full-length coding sequences SaCLCa2 and SaCLCc2 were amplified with primer pairs pMB1_SaCLCa2_F and pMB1_SaCLCa2_R, pMB1_SaCLCc2_F and pMB1_SaCLCc2_R ( Table S3 ), using the pVR2– SaCLCa2 and pVR2– SaCLCc2 constructs, respectively, as templates, and cloned in yeast expression vector pMB1 under control of a strong constitutive promoter GPD1 [ 60 ].…”

Section: Methodsmentioning

confidence: 99%

Chloride Channel Family in the Euhalophyte Suaeda altissima (L.) Pall: Cloning of Novel Members SaCLCa2 and SaCLCc2, General Characterization of the Family

Nedelyaeva¹,

Попова²,

Khramov³

et al. 2023

IJMS

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CLC family genes, comprising anion channels and anion/H+ antiporters, are widely represented in nearly all prokaryotes and eukaryotes. CLC proteins carry out a plethora of functions at the cellular level. Here the coding sequences of the SaCLCa2 and SaCLCc2 genes, homologous to Arabidopsis thaliana CLCa and CLCc, were cloned from the euhalophyte Suaeda altissima (L.) Pall. Both the genes cloned belong to the CLC family as supported by the presence of the key conserved motifs and glutamates inherent for CLC proteins. SaCLCa2 and SaCLCc2 were heterologously expressed in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encodes the only CLC family protein, the Cl– transporter Gef1p, in undisrupted strains of yeast. The Δgef1 strain is characterized by inability to grow on YPD yeast medium containing Mn2+ ions. Expression of SaCLCa2 in Δgef1 cells growing on this medium did not rescue the growth defect phenotype of the mutant. However, a partial growth restoration occurred when the Δgef1 strain was transformed by SaCLCa2(C544T), the gene encoding protein in which proline, specific for nitrate, was replaced with serine, specific for chloride, in the selectivity filter. Unlike SaCLCa2, expression of SaCLCc2 in Δgef1 resulted in a partial growth restoration under these conditions. Analysis of SaCLCa2 and SaCLCc2 expression in the euhalophyte Suaeda altissima (L.) Pall by quantitative real-time PCR (qRT-PCR) under different growth conditions demonstrated stimulation of SaCLCa2 expression by nitrate and stimulation of SaCLCc2 expression by chloride. The results of yeast complementation assay, the presence of both the “gating” and “proton” glutamates in aa sequences of both the proteins, as well results of the gene expression in euhalophyte Suaeda altissima (L.) Pall suggest that SaCLCa2 and SaCLCc2 function as anion/H+ antiporters with nitrate and chloride specificities, respectively. The general bioinformatic overview of seven CLC genes cloned from euhalophyte Suaeda altissima is given, together with results on their expression in roots and leaves under different levels of salinity.

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“…To carry out the knockout of the gene YNT1, the zeocin resistance gene (ZeoR) was incorporated into the YNT1 locus through homologous recombination at the YNT1 gene sites [44]. For this purpose, PCR fragments corresponding to "left" and "right" flanking parts of the YNT1 gene and the zeocin resistance gene ZeoR amplified from the pVR2 vector [57] were cloned into the bacterial vector pBlueScript KS(II)+ using Gibson assembly kit (NEB, Ipswich, MA, USA). "Left" and "Right" fragments of the YNT1 gene and the gene ZeoR were amplified using primer pairs ZeoYNTRfl_F and pBlueYNTRfl_R, pBlueYNTLfl_F and YNTLflZeo_R, and ZeoCas1 and ZeoCas2, respectively.…”

Section: Production Of a Deletion Mutant ∆Ynt1 Of H Polymorphamentioning

confidence: 99%

Molecular Cloning, Expression and Transport Activity of SaNPF6.3/SaNRT1.1, a Novel Protein of the Low-Affinity Nitrate Transporter Family from the Euhalophyte Suaeda altissima (L.) Pall.

Nedelyaeva,

Khramov,

Khalilova

et al. 2023

Membranes

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The SaNPF6.3 gene, a putative ortholog of the dual-affinity nitrate (NO3−) transporter gene AtNPF6.3/AtNRT1.1 from Arabidopsis thaliana, was cloned from the euhalophyte Suaeda altissima. The nitrate transporting activity of SaNPF6.3 was studied by heterologous expression of the gene in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the original nitrate transporter. Expression of SaNPF6.3 in Δynt1 cells rescued their ability to grow on the selective medium in the presence of nitrate and absorb nitrate from this medium. Confocal laser microscopy of the yeast cells expressing the fused protein GFP-SaNPF6.3 revealed GFP (green fluorescent protein) fluorescence localized predominantly in the cytoplasm and/or vacuoles. Apparently, in the heterologous expression system used, only a relatively small fraction of the GFP-SaNPF6.3 reached the plasma membrane of yeast cells. In S. altissima plants grown in media with either low (0.5 mM) or high (15 mM) NO3−; concentrations, SaNPF6.3 was expressed at various ontogenetic stages in different organs, with the highest expression levels in roots, pointing to an important role of SaNPF6.3 in nitrate uptake. SaNPF6.3 expression was induced in roots of nitrate-deprived plants in response to raising the nitrate concentration in the medium and was suppressed when the plants were transferred from sufficient nitrate to the lower concentration. When NaCl concentration in the nutrient solution was elevated, the SaNPF6.3 transcript abundance in the roots increased at the low nitrate concentration and decreased at the high one. We also determined nitrate and chloride concentrations in the xylem sap excreted by detached S. altissima roots as a function of their concentrations in the root medium. Based on a linear increase in Cl− concentrations in the xylem exudate as the external Cl− concentration increased and the results of SaNPF6.3 expression experiments, we hypothesize that SaNPF6.3 is involved in chloride transport along with nitrate transport in S. altissima plants.

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Biosynthesis of human β2-adrenergic receptor in methylotrophic yeast Pichia pastoris and its purification

Cited by 3 publications

References 34 publications

Molecular Cloning of Saslah3, a Novel Slow Anion Channel Slac/slah Family Gene, From Euhalophyte Suaeda Altissima (L.) Pall and Analysis of the Encoded Protein

Molecular Cloning of Saslah3, a Novel Slow Anion Channel Slac/slah Family Gene, From Euhalophyte Suaeda Altissima (L.) Pall and Analysis of the Encoded Protein

Chloride Channel Family in the Euhalophyte Suaeda altissima (L.) Pall: Cloning of Novel Members SaCLCa2 and SaCLCc2, General Characterization of the Family

Molecular Cloning, Expression and Transport Activity of SaNPF6.3/SaNRT1.1, a Novel Protein of the Low-Affinity Nitrate Transporter Family from the Euhalophyte Suaeda altissima (L.) Pall.

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