Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system
Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Wang, Ze [verfasserIn] |
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| Format: |
Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
1994 |
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| Schlagwörter: |
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| Anmerkung: |
© Chapman & Hall 1994 |
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| Übergeordnetes Werk: |
Enthalten in: Journal of muscle research and cell motility - Kluwer Academic Publishers, 1980, 15(1994), 6 vom: Dez., Seite 646-658 |
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| Übergeordnetes Werk: |
volume:15 ; year:1994 ; number:6 ; month:12 ; pages:646-658 |
| Links: |
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| DOI / URN: |
10.1007/BF00121072 |
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| Katalog-ID: |
OLC2067119362 |
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| 245 | 1 | 0 | |a Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system |
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| 500 | |a © Chapman & Hall 1994 | ||
| 520 | |a Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. | ||
| 650 | 4 | |a Electrophoretic Mobility | |
| 650 | 4 | |a Immunofluorescence Microscopy | |
| 650 | 4 | |a Harvest Time | |
| 650 | 4 | |a Carboxyl Terminus | |
| 650 | 4 | |a Heat Stability | |
| 700 | 1 | |a Horiuchi, Kurumi Y. |4 aut | |
| 700 | 1 | |a Jacob, Saji S. |4 aut | |
| 700 | 1 | |a Gopalakurup, Suresh |4 aut | |
| 700 | 1 | |a Chacko, Samuel |4 aut | |
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10.1007/BF00121072 doi (DE-627)OLC2067119362 (DE-He213)BF00121072-p DE-627 ger DE-627 rakwb eng 590 570 VZ 12 ssgn BIODIV DE-30 fid Wang, Ze verfasserin aut Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system 1994 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Chapman & Hall 1994 Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. Electrophoretic Mobility Immunofluorescence Microscopy Harvest Time Carboxyl Terminus Heat Stability Horiuchi, Kurumi Y. aut Jacob, Saji S. aut Gopalakurup, Suresh aut Chacko, Samuel aut Enthalten in Journal of muscle research and cell motility Kluwer Academic Publishers, 1980 15(1994), 6 vom: Dez., Seite 646-658 (DE-627)166717754 (DE-600)283053-X (DE-576)015170152 0142-4319 nnns volume:15 year:1994 number:6 month:12 pages:646-658 https://doi.org/10.1007/BF00121072 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-WIW GBV_ILN_2021 GBV_ILN_2219 GBV_ILN_2221 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4219 AR 15 1994 6 12 646-658 |
| spelling |
10.1007/BF00121072 doi (DE-627)OLC2067119362 (DE-He213)BF00121072-p DE-627 ger DE-627 rakwb eng 590 570 VZ 12 ssgn BIODIV DE-30 fid Wang, Ze verfasserin aut Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system 1994 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Chapman & Hall 1994 Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. Electrophoretic Mobility Immunofluorescence Microscopy Harvest Time Carboxyl Terminus Heat Stability Horiuchi, Kurumi Y. aut Jacob, Saji S. aut Gopalakurup, Suresh aut Chacko, Samuel aut Enthalten in Journal of muscle research and cell motility Kluwer Academic Publishers, 1980 15(1994), 6 vom: Dez., Seite 646-658 (DE-627)166717754 (DE-600)283053-X (DE-576)015170152 0142-4319 nnns volume:15 year:1994 number:6 month:12 pages:646-658 https://doi.org/10.1007/BF00121072 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-WIW GBV_ILN_2021 GBV_ILN_2219 GBV_ILN_2221 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4219 AR 15 1994 6 12 646-658 |
| allfields_unstemmed |
10.1007/BF00121072 doi (DE-627)OLC2067119362 (DE-He213)BF00121072-p DE-627 ger DE-627 rakwb eng 590 570 VZ 12 ssgn BIODIV DE-30 fid Wang, Ze verfasserin aut Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system 1994 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Chapman & Hall 1994 Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. Electrophoretic Mobility Immunofluorescence Microscopy Harvest Time Carboxyl Terminus Heat Stability Horiuchi, Kurumi Y. aut Jacob, Saji S. aut Gopalakurup, Suresh aut Chacko, Samuel aut Enthalten in Journal of muscle research and cell motility Kluwer Academic Publishers, 1980 15(1994), 6 vom: Dez., Seite 646-658 (DE-627)166717754 (DE-600)283053-X (DE-576)015170152 0142-4319 nnns volume:15 year:1994 number:6 month:12 pages:646-658 https://doi.org/10.1007/BF00121072 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-WIW GBV_ILN_2021 GBV_ILN_2219 GBV_ILN_2221 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4219 AR 15 1994 6 12 646-658 |
| allfieldsGer |
10.1007/BF00121072 doi (DE-627)OLC2067119362 (DE-He213)BF00121072-p DE-627 ger DE-627 rakwb eng 590 570 VZ 12 ssgn BIODIV DE-30 fid Wang, Ze verfasserin aut Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system 1994 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Chapman & Hall 1994 Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. Electrophoretic Mobility Immunofluorescence Microscopy Harvest Time Carboxyl Terminus Heat Stability Horiuchi, Kurumi Y. aut Jacob, Saji S. aut Gopalakurup, Suresh aut Chacko, Samuel aut Enthalten in Journal of muscle research and cell motility Kluwer Academic Publishers, 1980 15(1994), 6 vom: Dez., Seite 646-658 (DE-627)166717754 (DE-600)283053-X (DE-576)015170152 0142-4319 nnns volume:15 year:1994 number:6 month:12 pages:646-658 https://doi.org/10.1007/BF00121072 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-WIW GBV_ILN_2021 GBV_ILN_2219 GBV_ILN_2221 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4219 AR 15 1994 6 12 646-658 |
| allfieldsSound |
10.1007/BF00121072 doi (DE-627)OLC2067119362 (DE-He213)BF00121072-p DE-627 ger DE-627 rakwb eng 590 570 VZ 12 ssgn BIODIV DE-30 fid Wang, Ze verfasserin aut Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system 1994 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier © Chapman & Hall 1994 Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. Electrophoretic Mobility Immunofluorescence Microscopy Harvest Time Carboxyl Terminus Heat Stability Horiuchi, Kurumi Y. aut Jacob, Saji S. aut Gopalakurup, Suresh aut Chacko, Samuel aut Enthalten in Journal of muscle research and cell motility Kluwer Academic Publishers, 1980 15(1994), 6 vom: Dez., Seite 646-658 (DE-627)166717754 (DE-600)283053-X (DE-576)015170152 0142-4319 nnns volume:15 year:1994 number:6 month:12 pages:646-658 https://doi.org/10.1007/BF00121072 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_OLC FID-BIODIV SSG-OLC-WIW GBV_ILN_2021 GBV_ILN_2219 GBV_ILN_2221 GBV_ILN_4012 GBV_ILN_4082 GBV_ILN_4219 AR 15 1994 6 12 646-658 |
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English |
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Enthalten in Journal of muscle research and cell motility 15(1994), 6 vom: Dez., Seite 646-658 volume:15 year:1994 number:6 month:12 pages:646-658 |
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Enthalten in Journal of muscle research and cell motility 15(1994), 6 vom: Dez., Seite 646-658 volume:15 year:1994 number:6 month:12 pages:646-658 |
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Electrophoretic Mobility Immunofluorescence Microscopy Harvest Time Carboxyl Terminus Heat Stability |
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Journal of muscle research and cell motility |
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Wang, Ze @@aut@@ Horiuchi, Kurumi Y. @@aut@@ Jacob, Saji S. @@aut@@ Gopalakurup, Suresh @@aut@@ Chacko, Samuel @@aut@@ |
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Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. 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Wang, Ze |
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Wang, Ze ddc 590 ssgn 12 fid BIODIV misc Electrophoretic Mobility misc Immunofluorescence Microscopy misc Harvest Time misc Carboxyl Terminus misc Heat Stability Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system |
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Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system |
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Journal of muscle research and cell motility |
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overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system |
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Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system |
| abstract |
Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. © Chapman & Hall 1994 |
| abstractGer |
Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. © Chapman & Hall 1994 |
| abstract_unstemmed |
Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5′-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3′-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 μg per $ 10^{6} $ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of $ Ca^{2+} $-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant. © Chapman & Hall 1994 |
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