Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

Abstract

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over-expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step-wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys-rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 201

Development of a protocol to solidify native and artificial oil bodies for long‐term storage at room temperature

“…As expected, the molecular mass (27 kDa) of the recombinant caleosin was similar to that of the native caleosin extracted from sesame oil bodies (data not shown). Stable artificial oil bodies were successfully generated to form an emulsified solution in accord with our previous studies , …”

“…Artificial oil bodies were generated with 50 mg of sesame oil, 150 µg of dioleoyl phosphatidylcholine and 250 µg of recombinant caleosin in 1 mL of 10 mmol L −1 sodium phosphate buffer (pH 7.5) . Dioleoyl phosphatidylcholine dissolved in chloroform was placed at the bottom of an Eppendorf tube and the solvent was allowed to evaporate in a chemical hood overnight.…”

“…Artificial oil bodies were generated with 50 mg of sesame oil, 150 µg of dioleoyl phosphatidylcholine and 250 µg of recombinant caleosin in 1 mL of 10 mmol L −1 sodium phosphate buffer (pH 7.5). 21 Dioleoyl phosphatidylcholine dissolved in chloroform was placed at the bottom of an Eppendorf tube and the solvent was allowed to evaporate in a chemical hood overnight. After solvent evaporation, sesame oil and recombinant caleosin suspended in 1 mL of sodium phosphate buffer were incorporated, followed by sonication with a 3 mm diameter probe in a Sonics & Materials (Newtown, CT, USA) VCX750 ultrasonic processor with 30% amplification for 20 s, and samples were cooled in an ice bucket for 5 min.…”

Integral Proteins in Plant Oil Bodies

“…Recently, a system of generating antibodies against small molecules (haptens) was established under the assistance of artificial oil bodies [178]. To develop this system, a series of recombinant caleosins were engineered with more Lys residues to link and render small molecules on the surface of artificial oil bodies for antibody production.…”

N-terminus of seed caleosins is essential for lipid droplet sorting but not for lipid accumulation

“…They are widespread among species 42 from all kingdoms, in which they have general function or are asso- 43 ciated with special metabolism or living conditions. The composi- 44 tion of lipid droplets is simple: the highly hydrophobic lipid core is 45 surrounded by a phospholipid monolayer and associated proteins. 46 The number and type of stored lipids as well as the exact phospho- 47 lipids and proteins involved depend on the specific origin of lipid 48 droplets.…”