Here, we introduce a new type of molecular imprinted polymer (MIP) with immobilized assistant recognition polymer chains (ARPCs) to create effective recognition sites and with bacterial cloned protein as template for adsorbing the low content target protein from cell extract. In this work, cloned pig cyclophilin 18 (pCyP18), a peptidyl-prolyl cis/trans-isomerase, was used as template. The template protein was selectively assembled with ARPCs from their library, which consists of numerous limited length polymer chains with randomly distributed recognition sites of positively charged amino groups and immobilizing sites. These assemblies were adsorbed by porous microsphers and immobilized on them. After removing the template, binding sites complementary to the target protein in size, shape and the position of recognition groups were exposed, and their confirmation was preserved by the cross-linked structure. The synthesized MIP was used to adsorb the cellular pCyP18, and its proportional content was enriched more than hundred times. The extended experiment on imprinting bovine serum albumin (BSA) with ARPCs shows that this method is also suitable for large protein.
LONG Yi SUN Yang WANG Ying XING XiaoCui ZHAO Zhuo WANG ChunHong FAN YunGe MI HuaiFeng
We introduce a new method for separation/enrichment of the low-content cellular protein in high mo-lecular weight on the basis of molecular imprinting. The template protein, bacterial cloned immu-noglobulin binding protein (BiP), was selectively assembled with assistant recognition polymer chains (ARPCs) from their library, which consists of numerous limited length polymer chains with randomly distributed recognition and immobilizing sites. The assemblies of proteins and ARPCs were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After the template was removed, the synthesized imprinted polymer was used to adsorb authentic BiP from endoplasmic re-ticulum (ER) extract, and its proportional content was enriched 45 times. It is the first time that the low-content cellular natural protein, whose molecular weight reaches 78 kDa, is enriched by molecular imprinting.
A new protein molecularly imprinted polymer (MIP) was prepared with grafting polyvinyl alcohol as assistant recognition polymer chains (ARPCs). The ARPCs and acrylamide monomers were interpenetrated and then polymerized on the surface of macroporous acrylate adsorbent spheres. The template BSA was removed by treatment with 2.00 mol L-1 potassium chloride (KCl) solution and the adsorbed proteins were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 0.150, 0.500, and 2.00 mol L-1 KCl solutions were used as eluent to wash the adsorbed proteins. The SDS-PAGE results show that proteins washed out with 2.00 mol L-1KCl solution were from nonspecific adsorption of macroporous acrylate adsorbent spheres, and proteins washed out with 0.500 mol L-1KC1 solution were specific proteins imprinted by MIP resins. MIP resins with ARPCs had better recognition to the target proteins than that without ARPCs. The adsorption capacity of MIP resins immobilized ARPCs to the template BSA was about 80-100 μg g-1 when it was used for the adsorption of proteins mixture, and the specific adsorption of the target protein was obviously increased.
GUO MinJie1, GAO Ting1, FAN Zhi1, YAO JingXia1, XIA JianJun2 & MI HuaiFeng2 1Department of Chemistry, College of Sciences, Tianjin University of Science and Technology, Tianjin 300457, China
