Structure of recombinant human carboxylesterase 1 isolated from whole cabbage looper larvae
Harry M. Greenblatt, Tamara C. Otto, Melanie G. Kirkpatrick, Elena Kovaleva, Susan Brown, George Buchman, Douglas M. Cerasoli and Joel L. Sussman[1]
Molecular Tour
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. found in this space group, while the three twofold axes at z = 1/4 that intersect on this axis complete the . An gave an r.m.s. deviation of 0.42 Å for 522 Cα atoms (2h7c is colored in red and rhCES1 is in green). An r.m.s. value of 0.47 Å (3132 Cα atoms) was obtained for the , 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 . The poorly defined density for this same region in rhCES1 is consistent with the observation that (the loop of one subunit is in red and the loop of the second subunit is in orange).
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.
References
- ↑ 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
