Journal:Acta Cryst F:S1744309112003326
From Proteopedia
(Difference between revisions)
| Line 5: | Line 5: | ||
<b>Molecular Tour</b><br> | <b>Molecular Tour</b><br> | ||
| - | The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 (rhCES1) has been produced in and isolated from whole ''Trichoplusia ni'' larvae. The recombinant protein was crystallized and its structure was solved to 2.2 Å resolution. The current structure of rhCES1 represents the first published hexagonal crystal form, despite the fact that all other published examples of hCES1 structures consist of a hexamer in the asymmetric unit. <scene name='Journal:Acta_Cryst_F:1/Cv/4'>The trimer of subunits sits around one of the threefold axes</scene> found in this space group, while the three twofold axes at z = 1/4 that intersect on this axis complete the <scene name='Journal:Acta_Cryst_F:1/Cv/5'>hexamer</scene>. An <scene name='Journal:Acta_Cryst_F:1/Cv/6'>alignment of the A chain from PDB entry 2h7c with the asymmetric unit reported here</scene> gave an r.m.s. deviation of 0.42 Å for 522 Cα atoms ([[2h7c]] <font color='red'><b>is colored in red</b></font> and <span style="color:lime;background-color:black;font-weight:bold;">rhCES1 is in green</span>). An r.m.s. value of 0.47 Å (3132 Cα atoms) was obtained for the <scene name='Journal:Acta_Cryst_F:1/Cv/7'>entire 2h7c hexamer superposed with the symmetry-generated rhCES1 hexamer</scene>, indicating that the quaternary structure is essentially identical in these crystal forms isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies. | + | The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 (rhCES1) has been produced in and isolated from whole ''Trichoplusia ni'' larvae. The recombinant protein was crystallized and its structure was solved to 2.2 Å resolution. The current structure of rhCES1 ([[4ab1]]) represents the first published hexagonal crystal form, despite the fact that all other published examples of hCES1 structures consist of a hexamer in the asymmetric unit. <scene name='Journal:Acta_Cryst_F:1/Cv/4'>The trimer of subunits sits around one of the threefold axes</scene> found in this space group, while the three twofold axes at z = 1/4 that intersect on this axis complete the <scene name='Journal:Acta_Cryst_F:1/Cv/5'>hexamer</scene>. An <scene name='Journal:Acta_Cryst_F:1/Cv/6'>alignment of the A chain from PDB entry 2h7c with the asymmetric unit reported here</scene> gave an r.m.s. deviation of 0.42 Å for 522 Cα atoms ([[2h7c]] <font color='red'><b>is colored in red</b></font> and <span style="color:lime;background-color:black;font-weight:bold;">rhCES1 is in green</span>). An r.m.s. value of 0.47 Å (3132 Cα atoms) was obtained for the <scene name='Journal:Acta_Cryst_F:1/Cv/7'>entire 2h7c hexamer superposed with the symmetry-generated rhCES1 hexamer</scene>, indicating that the quaternary structure is essentially identical in these crystal forms isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies. |
Regions of the current structure that differ from the previously reported examples of hCES1 include Ser365–Asp374, which has very poor density. In the [[2h7c]] structure <scene name='Journal:Acta_Cryst_F:1/Cv/12'>each of the six examples of the section of chain from Ser365 to Asp374 has a different conformation</scene>. The poorly defined density for this same region in rhCES1 is consistent with the observation that <scene name='Journal:Acta_Cryst_F:1/Cv/13'>this loop can adopt multiple conformations despite its participation in forming the twofold interface between chains</scene> (<font color='red'><b>the loop of one subunit is in red</b></font> and <span style="color:orange;background-color:black;font-weight:bold;">the loop of the second subunit is in orange</span>). | Regions of the current structure that differ from the previously reported examples of hCES1 include Ser365–Asp374, which has very poor density. In the [[2h7c]] structure <scene name='Journal:Acta_Cryst_F:1/Cv/12'>each of the six examples of the section of chain from Ser365 to Asp374 has a different conformation</scene>. The poorly defined density for this same region in rhCES1 is consistent with the observation that <scene name='Journal:Acta_Cryst_F:1/Cv/13'>this loop can adopt multiple conformations despite its participation in forming the twofold interface between chains</scene> (<font color='red'><b>the loop of one subunit is in red</b></font> and <span style="color:orange;background-color:black;font-weight:bold;">the loop of the second subunit is in orange</span>). | ||
The current results confirm that rhHCES1 isolated from the ''T. ni'' system is essentially identical to previous examples of this enzyme isolated from cultured insect cells, validating the use of the whole insect system as a source for recombinant proteins in structure determination studies. | The current results confirm that rhHCES1 isolated from the ''T. ni'' system is essentially identical to previous examples of this enzyme isolated from cultured insect cells, validating the use of the whole insect system as a source for recombinant proteins in structure determination studies. | ||
Revision as of 11:50, 16 January 2013
| |||||||||||
- ↑ Greenblatt HM, Otto TC, Kirkpatrick MG, Kovaleva E, Brown S, Buchman G, Cerasoli DM, Sussman JL. Structure of recombinant human carboxylesterase 1 isolated from whole cabbage looper larvae. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Mar 1;68(Pt 3):269-72., Epub 2012 Feb 15. PMID:22442219 doi:10.1107/S1744309112003326
This page complements a publication in scientific journals and is one of the Proteopedia's Interactive 3D Complement pages. For aditional details please see I3DC.
