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Article: High-level expression of recombinant Aplysia ADP-ribosyl cyclase in Pichia pastoris by fermentation

TitleHigh-level expression of recombinant Aplysia ADP-ribosyl cyclase in Pichia pastoris by fermentation
Authors
Issue Date1997
PublisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/yprep
Citation
Protein Expression And Purification, 1997, v. 11 n. 1, p. 104-110 How to Cite?
AbstractCyclic ADP-ribose (cADPR), a Ca2+ mobilizing cyclic nucleotide derived from NAD+, is rapidly emerging as an endogenous modulator of Ca2+-induced Ca2+ release mechanisms in various cellular systems. ADP ribosyl cyclase, first isolated from the marine invertebrate Aplysia californica, cyclizes NAD+ to cADPR. In this study we have utilized the methylotrophic yeast Pichia pastoris to express high levels of this enzyme. The cyclase construct consisted of the soluble domain, with isoleucine (25 residues following the initial methionine) as the N-terminus, cloned in frame with the yeast α-factor mating signal sequence. Cyclase yeast transformants were screened using the Zeocin (phleomycin from Streptomyces verticillus) selectable marker which resulted in 100% active transformation. All active clones comprised the methanol utilization slow (Mut8) phenotype. The protein was expressed using the tightly regulated methanol-inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae α-factor mating secretion signal. Using high biomass fermentations, up to 300 mg/liter of cyclase was achieved. SDS-PAGE analysis revealed that the heterologous protein comprised nearly 90-95% of the total protein secreted extracellularly. The enzyme characteristics of the recombinant cyclase compared favorably with those of the native enzyme. The yeast expression system can thus produce gram quantities of this novel protein.
Persistent Identifierhttp://hdl.handle.net/10722/171642
ISSN
2015 Impact Factor: 1.407
2015 SCImago Journal Rankings: 0.767
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorMunshi, Cen_US
dc.contributor.authorHon, CLen_US
dc.date.accessioned2012-10-30T06:16:06Z-
dc.date.available2012-10-30T06:16:06Z-
dc.date.issued1997en_US
dc.identifier.citationProtein Expression And Purification, 1997, v. 11 n. 1, p. 104-110en_US
dc.identifier.issn1046-5928en_US
dc.identifier.urihttp://hdl.handle.net/10722/171642-
dc.description.abstractCyclic ADP-ribose (cADPR), a Ca2+ mobilizing cyclic nucleotide derived from NAD+, is rapidly emerging as an endogenous modulator of Ca2+-induced Ca2+ release mechanisms in various cellular systems. ADP ribosyl cyclase, first isolated from the marine invertebrate Aplysia californica, cyclizes NAD+ to cADPR. In this study we have utilized the methylotrophic yeast Pichia pastoris to express high levels of this enzyme. The cyclase construct consisted of the soluble domain, with isoleucine (25 residues following the initial methionine) as the N-terminus, cloned in frame with the yeast α-factor mating signal sequence. Cyclase yeast transformants were screened using the Zeocin (phleomycin from Streptomyces verticillus) selectable marker which resulted in 100% active transformation. All active clones comprised the methanol utilization slow (Mut8) phenotype. The protein was expressed using the tightly regulated methanol-inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae α-factor mating secretion signal. Using high biomass fermentations, up to 300 mg/liter of cyclase was achieved. SDS-PAGE analysis revealed that the heterologous protein comprised nearly 90-95% of the total protein secreted extracellularly. The enzyme characteristics of the recombinant cyclase compared favorably with those of the native enzyme. The yeast expression system can thus produce gram quantities of this novel protein.en_US
dc.languageengen_US
dc.publisherAcademic Press. The Journal's web site is located at http://www.elsevier.com/locate/yprepen_US
dc.relation.ispartofProtein Expression and Purificationen_US
dc.subject.meshAdp-Ribosyl Cyclaseen_US
dc.subject.meshAnimalsen_US
dc.subject.meshAntigens, Cd - Biosynthesis - Genetics - Isolation & Purificationen_US
dc.subject.meshAntigens, Cd38en_US
dc.subject.meshAntigens, Differentiation - Biosynthesis - Genetics - Isolation & Purificationen_US
dc.subject.meshAplysia - Enzymologyen_US
dc.subject.meshChromatography, Ion Exchangeen_US
dc.subject.meshFermentationen_US
dc.subject.meshFluorometryen_US
dc.subject.meshMultienzyme Complexes - Biosynthesis - Genetics - Isolation & Purificationen_US
dc.subject.meshNad+ Nucleosidase - Biosynthesis - Genetics - Isolation & Purificationen_US
dc.subject.meshPichiaen_US
dc.subject.meshRecombinant Proteins - Biosynthesis - Isolation & Purificationen_US
dc.titleHigh-level expression of recombinant Aplysia ADP-ribosyl cyclase in Pichia pastoris by fermentationen_US
dc.typeArticleen_US
dc.identifier.emailHon, CL:leehc@hku.hken_US
dc.identifier.authorityHon, CL=rp00545en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1006/prep.1997.0773en_US
dc.identifier.pmid9325145-
dc.identifier.scopuseid_2-s2.0-0031260305en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0031260305&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume11en_US
dc.identifier.issue1en_US
dc.identifier.spage104en_US
dc.identifier.epage110en_US
dc.identifier.isiWOS:A1997XY80200013-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridMunshi, C=7003972383en_US
dc.identifier.scopusauthoridHon, CL=26642959100en_US

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