Method For Control The Orientation Of Proteins Immobilization On Solid Surface

Coupling technique of protein on solid phase surface is widely used in the field of bioseparation, enzyme catalysis, disease diagnosis and analytical testing. The spatial orientation of protein on solid phase surface is an important impact factor on the performance of separation materials, enzyme catalytic activity and detection accuracy and sensitivity. Due to the complexity of protein structure, there are some differences on surface charge and hydrophilicity in different condition. Congtrolling the spatial orientation of protein on solid surface exist some difficulties. In the present research, the method of identifying the coupling sites of model proteins on the agarose gel was studied by HPLC-MS and the method of proteolysis. On this basis, then established a new biomacromolecule coupling technology based on click chemistry, which was controllable to the spatial orientation on the surface of solid phase. The main contents are as follows:(1) A method for site-directed coupling of protein on sepharose microspheres was developed using click chemistry with lysozyme as model protein. Sepharose microspheres was activated by 1,4-butanediol diglycidyl ether, and alkynyl group was introduced by reaction of the epoxy group with 4-ethynlaniline. Azido-decanoic acid was synthesized and used to modify lysozyme by EDC/NHS method. The modified lysozymes was separated using chromatographic method and three isomers with different modification sites were obtained. Three azido-decanoic-linking sites in three isomers were identified as Lys1, Lys33 and Lys96 respectively using HPLC-MS. The azido-decanoic acid-modified lysozymes with Lys96 as linking site was separated and further reacted with 4-ethynlaniline-modified sepharose microspheres. The tryptic digest of the coupled lysozyme was analyzed by HPLC-MS, and the Gln74-Lys96 was disappeared, indicated that link site of lysozyme on sepharose microspheres was Lys96.(2) Staphylococcal Protein A(SpA) was selected as model protein, and used HPLC-MS and method of proteolysis to identify the coupling sites of protein on the Sepharose 4FF. The results indicated that K99、K447、K459and K470 were the coupling sites of SpA under the condition of 10.5 pH and 20.21 μmol/g epoxy group density of agarose surface, and the proportions of these coupling sites are 99%,72%、87% and 97% respectively. It is demonstrated that the method of proteolysis combining the peptide sequence analysis by MS could identify the coupling sites of protein on affinity medium effectively.A method for site-direct coupling of protein on sepharose microspheres was developed using click chemistry. The azido-decanoic acid-modified lysozymes was separated and further reacted with 4-ethynlaniline-modified sepharose microspheres. the method for site-direct coupling of lysozyme on sepharose microspheres using click chemistry was confirmed. HPLC-MS and enzymatic digestion can be used to identify the coupling sites of protein on the media.