ABSTRACT
Stable oil bodies of smaller sizes and higher thermostability were isolated from mature cycad (Cycas revoluta) megagametophytes compared with those isolated from sesame seeds. Immunological cross-recognition revealed that cycad oil bodies contained a major protein of 27 kDa, tentatively identified as caleosin, while oleosin, the well-known structural protein, was apparently absent. Mass spectrometric analysis showed that the putative cycad caleosin possessed a tryptic fragment of 15 residues matching to that of a theoretical moss caleosin. A complete cDNA fragment encoding this putative caleosin was obtained by PCR cloning using a primer designed according to the tryptic peptide and another one designed according to a highly conservative region among diverse caleosins. The identification of this clone was subsequently confirmed by immunodetection and MALDI-MS analyses of its recombinant fusion protein over-expressed in Escherichia coli and the native form from cycad oil bodies. Stable artificial oil bodies were successfully constituted with triacylglycerol, phospholipid and the recombinant fusion protein containing the cycad caleosin. These results suggest that stable oil bodies in cycad megagametophytes are mainly sheltered by a unique structural protein caleosin.
Subject(s)
Calcium-Binding Proteins/metabolism , Cycadopsida/metabolism , Organelles/metabolism , Plant Oils/metabolism , Plant Proteins/metabolism , Amino Acid Sequence , Calcium-Binding Proteins/genetics , Cloning, Molecular , Conserved Sequence , Cycadopsida/genetics , DNA, Complementary/isolation & purification , Escherichia coli/metabolism , Mass Spectrometry , Molecular Sequence Data , Phospholipids , Plant Oils/chemistry , Plant Oils/isolation & purification , Plant Proteins/genetics , Polymerase Chain Reaction , RNA, Plant/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , TriglyceridesABSTRACT
Pectin methylesterase (PME) in jelly fig (Ficus awkeotsang) achenes is an N-glycosylated enzyme responsible for the gelation of jelly curd. A recombinant jelly fig PME was overexpressed in Escherichia coli and confirmed by immunodetection and LC-nanoESI-MS/MS analysis. To identify the N-glycosylation site, native PME and its deglycosylated and recombinant forms, which lacked glycan, were purified and subjected to comparative MALDI-MS mapping of the corresponding tryptic fragments. The results showed that N-glycosylation occurred at Asn(153) of the mature jelly fig PME, the only glycosylation site predicted by its sequence analysis. The major N-glycans released from the native PME by PNGase F were identified by MS/MS analyses as xylosylated, noncore fucosylated pauci-mannose, and complex-type structures. Molecular modeling of the 3D structure of jelly fig PME indicated that the N-glycan was putatively attached to the back region of the active site of this enzyme.
Subject(s)
Carboxylic Ester Hydrolases/chemistry , Ficus/enzymology , Plant Proteins/chemistry , Polysaccharides/chemistry , Amino Acid Sequence , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/metabolism , Ficus/chemistry , Ficus/genetics , Glycosylation , Molecular Sequence Data , Plant Proteins/genetics , Plant Proteins/metabolism , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.