Production of native creatine kinase B in insect cells using a baculovirus expression vector

Yvette J.M. de Kok; Monique P.A. Geurds; Erik A. Sistermans; Magda Usmany; Just M. Vlak; Bé Wieringa

doi:10.1007/BF00925927

Production of native creatine kinase B in insect cells using a baculovirus expression vector

Yvette J.M. de Kok^*, Monique P.A. Geurds, Erik A. Sistermans, Magda Usmany, Just M. Vlak, Bé Wieringa

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.

Original language	English
Pages (from-to)	59-65
Number of pages	7
Journal	Molecular and Cellular Biochemistry
Volume	143
Issue number	1
DOIs	https://doi.org/10.1007/BF00925927
Publication status	Published - Feb 1995

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title = "Production of native creatine kinase B in insect cells using a baculovirus expression vector",

abstract = "A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.",

keywords = "baculovirus, creatine kinase B, monoclonal antibody, SF9 cells",

author = "{de Kok}, {Yvette J.M.} and Geurds, {Monique P.A.} and Sistermans, {Erik A.} and Magda Usmany and Vlak, {Just M.} and B{\'e} Wieringa",

year = "1995",

month = feb,

doi = "10.1007/BF00925927",

language = "English",

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T1 - Production of native creatine kinase B in insect cells using a baculovirus expression vector

AU - de Kok, Yvette J.M.

AU - Geurds, Monique P.A.

AU - Sistermans, Erik A.

AU - Usmany, Magda

AU - Vlak, Just M.

AU - Wieringa, Bé

PY - 1995/2

Y1 - 1995/2

N2 - A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.

AB - A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.

KW - baculovirus

KW - creatine kinase B

KW - monoclonal antibody

KW - SF9 cells

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JO - Molecular and Cellular Biochemistry

JF - Molecular and Cellular Biochemistry

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ER -

Production of native creatine kinase B in insect cells using a baculovirus expression vector

Abstract

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