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001-es BibID:BIBFORM105430
035-os BibID:(cikkazonosító)1215 (WoS)000895364600001
Első szerző:Borrego, Jesús
Cím:Recombinant Expression in Pichia pastoris System of Three Potent Kv1.3 Channel Blockers : Vm24, Anuroctoxin, and Ts6 / Borrego Jesús, Naseem Muhammad Umair, Sehgal Al Nasar Ahmed, Panda Lipsa Rani, Shakeel Kashmala, Gaspar Attila, Nagy Cynthia, Varga Zoltan, Panyi Gyorgy
Dátum:2022
ISSN:2309-608X
Megjegyzések:The Kv1.3 channel has become a therapeutic target for the treatment of various diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom; however, extensive studies require amounts of toxin that cannot be readily obtained directly from venoms. The Pichia pastoris expression system provides a cost-effective approach to overcoming the limitations of chemical synthesis and E. coli recombinant expression. In this work, we developed an efficient system for the production of three potent Kv1.3 channel blockers from different scorpion venoms: Vm24, AnTx, and Ts6. Using the Pichia system, these toxins could be obtained in sufficient quantities (Vm24 1.6 mg/L, AnTx 46 mg/L, and Ts6 7.5 mg/L) to characterize their biological activity. A comparison was made between the activity of tagged and untagged recombinant peptides. Tagged Vm24 and untagged AnTx are nearly equivalent to native toxins in blocking Kv1.3 (Kd = 4.4 pM and Kd = 0.72 nM, respectively), whereas untagged Ts6 exhibits a 53-fold increase in Kd (Kd = 29.1 nM) as compared to the native peptide. The approach described here provides a method that can be optimized for toxin production to develop more selective and effective Kv1.3 blockers with therapeutic potential.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Pichia pastoris
recombinant peptides
scorpion venoms
Kv1.3 blockers
Megjelenés:Journal of Fungi. - 8 : 11 (2022), p. 1-15. -
További szerzők:Naseem, Muhammad Umair (1993-) (biofizikus, molekuláris biológus) Sehgal, Al Nasar Ahmed Panda, Lipsa Rani Shakeel, Kashmala Gáspár Attila (1970-) (vegyész, kémikus) Nagy Cynthia (1994-) Varga Zoltán (1969-) (biofizikus, szakfordító) Panyi György (1966-) (biofizikus)
Pályázati támogatás:K143071
OTKA
K127931
OTKA
Stipendium Hungaricum Scholarship by the Tempus Public Foundation
Egyéb
Richter Gedeon Talentum Foundation
Egyéb
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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2.

001-es BibID:BIBFORM098765
035-os BibID:(cikkazonosító)733610 (scopus)85117131062 (wos)000707249000001
Első szerző:Naseem, Muhammad Umair (biofizikus, molekuláris biológus)
Cím:Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin / Naseem Muhammad Umair, Tajti Gabor, Gaspar Attila, Szanto Tibor G., Borrego Jesús, Panyi Gyorgy
Dátum:2021
ISSN:1663-9812
Megjegyzések:Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1?Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ? 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.
Tárgyszavak:Orvostudományok Gyógyszerészeti tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Pichia pastoris
patch-clamp
margatoxin
recombinant expression
Kv1.3 blocker
CD4+ TEM cells
Megjelenés:Frontiers in Pharmacology. - 12 (2021), p. 1-18. -
További szerzők:Tajti Gábor (1988-) (gyógyszerész, biofizikus, sejtbiológus) Gáspár Attila (1970-) (vegyész, kémikus) Szántó Gábor Tibor (1980-) (vegyész) Borrego, Jesús Panyi György (1966-) (biofizikus)
Pályázati támogatás:K119417
OTKA
EFOP-3.6.1-16-2016-00022
EFOP
GINOP2.3.2-15-2016-00044
GINOP
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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