Molecular imprinted polymer with positively charged assistant recognition polymer chains for adsorption/enrichment of low content target protein

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.

[1]  J. Niu,et al.  Reversible disulfide cross-linking in layer-by-layer films: preassembly enhanced loading and pH/reductant dually controllable release. , 2007, Langmuir : the ACS journal of surfaces and colloids.

[2]  G. Wulff,et al.  Enzyme-like catalysis by molecularly imprinted polymers. , 2002, Chemical reviews.

[3]  F. Shi,et al.  Surface Imprinting in Layer‐by‐Layer Nanostructured Films , 2007 .

[4]  Klaus Mosbach,et al.  Drug assay using antibody mimics made by molecular imprinting , 1993, Nature.

[5]  K. Lam,et al.  An Unnatural Amino Acid that Induces β-Sheet Folding and Interaction in Peptides , 2002 .

[6]  K. Shea,et al.  Selective protein capture by epitope imprinting. , 2006, Angewandte Chemie.

[7]  Karsten Haupt,et al.  Creating a good impression , 2002, Nature Biotechnology.

[8]  Stellan Hjertén,et al.  Gels mimicking antibodies in their selective recognition of proteins , 1997 .

[9]  D. Speicher,et al.  Cyclophilin: a specific cytosolic binding protein for cyclosporin A. , 1984, Science.

[10]  B. Bowman,et al.  Isolation and characterization of the Neurospora crassa endoplasmic reticulum , 1983, Journal of bacteriology.

[11]  Min Guo,et al.  Protein-imprinted polymer with immobilized assistant recognition polymer chains. , 2006, Biomaterials.

[12]  N. Minoura,et al.  Synthesis of Polymer-Coated Silica Particles with Specific Recognition Sites for Glucose Oxidase by the Molecular Imprinting Technique. , 1998 .

[13]  T. Guo,et al.  Adsorptive separation of hemoglobin by molecularly imprinted chitosan beads. , 2004, Biomaterials.

[14]  D L Venton,et al.  Influence of protein on polysiloxane polymer formation: evidence for induction of complementary protein-polymer interactions. , 1995, Biochimica et biophysica acta.

[15]  K. Mizuno,et al.  PHOTO-INDUCED CYCLODIMERIZATION OF ELECTRON-RICH AROMATIC OLEFINS IN THE PRESENCE OF TRANSITION METAL COMPLEXES , 1979 .

[16]  N. Minoura,et al.  Molecular imprinting: synthesis of polymer particles with antibody-like binding characteristics for glucose oxidase. , 1996, Biochemical and biophysical research communications.

[17]  Hui Li,et al.  The mouse FKBP23 binds to BiP in ER and the binding of C‐terminal domain is interrelated with Ca2+ concentration , 2004, FEBS letters.

[18]  Husheng Yan,et al.  Atom transfer radical polymerization of solketal acrylate using cyclohexanone as the solvent , 2005 .

[19]  Buddy D. Ratner,et al.  Template-imprinted nanostructured surfaces for protein recognition , 1999, Nature.

[20]  J. Liu,et al.  Crystal structure of recombinant human T-cell cyclophilin A at 2.5 A resolution. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. Rachkov,et al.  Recognition of oxytocin and oxytocin-related peptides in aqueous media using a molecularly imprinted polymer synthesized by the epitope approach. , 2000, Journal of chromatography. A.

[22]  A. Turner,et al.  Surface-grafted molecularly imprinted polymers for protein recognition. , 2001, Analytical chemistry.

[23]  G. Wulff.  Molecular Imprinting in Cross‐Linked Materials with the Aid of Molecular Templates— A Way towards Artificial Antibodies , 1995 .

[24]  Klaus Mosbach,et al.  An approach towards surface imprinting using the enzyme ribonuclease A , 1995, Journal of molecular recognition : JMR.

[25]  Yun-ge Fan,et al.  Molecular imprinted polymer with cloned bacterial protein template enriches authentic target in cell extract , 2006, FEBS letters.

[26]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[27]  B. Sellergren,et al.  Imprinting of amino acid derivatives in macroporous polymers , 1984 .

[28]  Huai-feng Mi,et al.  Binding of FKBP23 to BiP in ER Shown by Gel Filtration Chromatography , 2007, Zeitschrift fur Naturforschung. C, Journal of biosciences.

[29]  H. Gross,et al.  Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels , 1987 .

[30]  Di Wu,et al.  The binding of FKBP23 to BiP modulates BiP's ATPase activity with its PPIase activity. , 2007, Biochemical and biophysical research communications.