Immunogenic molecules
An immunogenic and molecular technology, applied in the field of immunology, can solve problems such as ineffectiveness and ineffectiveness of full-length proteins
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Embodiment 1
[0182] Materials and Methods
[0183] Reagent
[0184] Unless otherwise specified, chemical reagents are of analytical grade or equivalent. N,N'-dimethylamide (DMF), piperidine, trifluoroacetic acid (TFA), O'-benzotriazole-N,N,N',N'-tetramethylurea cationic hexafluorophosphate (HBTU), 1-Hydroxybenzotriazole (HOBt), Diisopropylethylamine (DIPEA) and Diisopropylcarbodiimide (DIPCDI) were from Auspep Pty in Melbourne, Australia and Sigma- Aldrich Pty Company. Dichloromethane (DCM) and diethyl ether were purchased from MerckPty (Kilsyth, Australia). Phenol and triisopropylsilane (TIPS) are from Aldrich (Milwaulke, WI), trinitrobenzene methanesulfonic acid (TNBSA) and diaminopyrimidine (DMAP) are from Fluka; 1,8-diazabicyclo[ 5.4.0] Undec-7-ene (DBU) is from Sigma, palmitic acid is from Fluka. The solid supports TentaGel S RAM and TentaGel S Am were from Rapp Polymere GmbH, Tubingen, Germany. O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethylene glycol (Fmoc-PEG) was obtained from...
Embodiment 2
[0210] Synthesis of four different types of lipidated HEL (lysozyme) proteins
[0211] Four different lipidated HELs were prepared by coupling the four lipid moieties listed in Figure 1 to the egg lysozyme (HEL) protein. Figure 3 shows a schematic diagram of these four lipidated proteins.
[0212]Lipidation 1HEL (thioether) and lipidation in lipidated HEL protein 2 HEL (thioether) is prepared by derivatizing HEL with MCS and then chemically selectively connecting the sulfhydryl group of structure A to form a thioether bond between the protein and the lipid assembly. Their difference lies in lipidation 2 HEL (thioether) has two copies of structure A. Lipidation of branches 2 HEL has only one copy of lipid component per protein molecule, but because of the bivalent nature of structure B, there are two copies of pam2cys per protein. This makes it more hydrophobic and elution by HPLC is much delayed.
[0213] To prepare lipidation 1 HEL (disulfide), HEL is modified with iso(yl) bifunc...
Embodiment 3
[0215] Evaluation of the immunogenicity of lipidated insulin
[0216] The insulin is lipidated by the following procedure. 10mg of bovine insulin was dissolved in 400μl of 0.5M phosphate buffer (pH 7.9) containing 6M guanidine hydrochloride and 400μl of 0.02M phosphate buffer (pH 7). 200μl of acetonitrile solution containing 3.25mg of N-succinimidyl 6-maleimidohexanoate (MCS) was added to this solution. After 3 hours of action, the MCS-modified insulin was treated by semi-preparative HPLC purification. 3.3 mg MCS modified insulin and 5 mg Pam2CysSer(Lys)8Cys were dissolved in 500 μl acetonitrile and 500 μl water. The reaction mixture was left at room temperature for 48 hours. Two lipidated insulin compounds were separated by semi-preparative HPLC. Using mass spectrometry to analyze these components showed that two different lipidated insulins were obtained, and the number of lipid moieties attached to each protein molecule was different. Pam2Cys 2 -Insulin has two copies of Pam pe...
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