title: Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system.
abstract: Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were glycosylated, enzymatically active, and had an apparent molecular weight similar to native hGL (approx. 50kDa). The recombinant hGLs were stable under acidic conditions and in the presence of gastric pepsin. Moreover, as found with the naturally occurring enzyme, the activity of recombinant hGL on the short chain lipid, tributyrin, was higher than on long chain Intralipid 30% emulsion. The maximum specific activity measured on tributyrin was 310 U/mg of protein and the maximum yield was 193 U/g of infiltrated leaf tissue. These results show that transient expression in plants can be used to produce active hGL that could be efficiently purified using established techniques. The approach provides a means of generating large quantities of hGL that could be of use for a number of applications both in vitro and in vivo.
authors.affiliation: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Vardakou
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
authors.forename: Frank
authors.initials: F
authors.lastname: Sainsbury
authors.affiliation: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Rigby
authors.affiliation: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.
authors.forename: Francis
authors.initials: F
authors.lastname: Mulholland
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK. Electronic address: george.lomonossoff@jic.ac.uk.
authors.forename: George P
authors.initials: GP
authors.lastname: Lomonossoff
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D017798:Comovirus / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
chemical_list: D006639:Histidine; D006923:Hymecromone; D008049:Lipase; D011993:Recombinant Fusion Proteins; C023520:4-methylumbelliferyl oleate; C485982:LIPF protein, human