Macroporous molecularly imprinted monolithic polymer columns for protein recognition by liquid chromatography.

Macroporous cytochrome c (cyc)-imprinted monolithic polyarylamide columns were prepared, and applied for the template protein recognition by HPLC. With cyc (18.8 mg) as template, the imprinted monolithic materials were in situ polymerized in an HPLC column tube, with methacrylamide (450 mg), methacrylic acid (15.8 mg), piperazine diacrylamide (720 mg) and ammonium sulfate (390 mg) dissolved in 5 mL of phosphate buffer (pH 7.4), initiated by ammonium persulfate and TEMED. After the reaction, cyc was removed with acetic acid (10%, v/v) containing 10% w/v SDS. The non-imprinted monolithic column was prepared under the same procedure except without cyc. Retention of cyc and its competitive protein, lysozyme (lys), on molecular-imprinting polymer (MIP) and non-imprinted polymer columns was studied. When the pH value of mobile phase was 4.0, on MIP column, the retention factors of cyc and lys were 2.0 and 1.3, respectively. However, those on non-imprinted polymer column were very similar, both as 1.1. Even in competitive environment, a mixture of cyc and lys could be separated on MIP column under gradient elution, with resolution as 1.2. These results indicate that protein-imprinted monolithic polymer columns could offer obvious affinity and specific recognition to the template protein.

The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus).

Sperm-mediated gene transfer (SMGT) is a promising transgenic technology that relies on the capability of sperm to internalize exogenous DNA. In marine fish, however, the interaction between sperm and exogenous DNA appears to be deficient. Here, we demonstrated significant DNase activity in the seminal plasma of the olive flounder. When incubated with naked-DNA, the spermatozoa lost their structural integrity, including the head, mitochondria and flagellum, in an incubation time-dependent manner. However, internalization of a liposome-DNA complex resulted in the structural integrity of the spermatozoa being maintained, even when using incubation times of up to 50min. We concluded that in the olive flounder, SMGT is possible by integrating liposome-DNA complexes, rather than naked-DNA alone, into the sperm. In brief, removal of the seminal plasma and packaging the exogenous DNA were necessary for successful SMGT in the olive flounder.

Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum.

Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g(-1). And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.