Surface imprinted bio‐nanocomposites for affinity separation of a cellular DNA repair protein

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional DNA repair protein localized in different subcellular compartments. The mechanisms responsible for the highly regulated subcellular localization and “interactomes” of this protein are not fully understood but have been closely correlated to the posttranslational modifications in different biological context. In this work, we attempted to develop a bio‐nanocomposite with antibody‐like properties that could capture APE1 from cellular matrices to enable the comprehensive study of this protein. By fixing the template APE1 on the avidin‐modified surface of silica‐coated magnetic nanoparticles, we first added 3‐aminophenylboronic acid to react with the glycosyl residues of avidin, followed by addition of 2‐acrylamido‐2‐methylpropane sulfonic acid as the second functional monomer to perform the first step imprinting reaction. To further enhance the affinity and selectivity of the binding sites, we carried out the second step imprinting reaction with dopamine as the functional monomer. After the polymerization, we modified the nonimprinted sites with methoxypoly (ethylene glycol) amine (mPEG‐NH2). The resulting molecularly imprinted polymer‐based bio‐nanocomposite showed high affinity, specificity, and capacity for template APE1. It allowed for the extraction of APE1 from the cell lysates with high recovery and purity. Moreover, the bound protein could be effectively released from the bio‐nanocomposite with high activity. The bio‐nanocomposite offers a very useful tool for the separation of APE1 from various complex biological samples.

Exploring the Complex Impact of Proteins on Dopamine Polymerization: Mechanisms and Strategies for Modulation

“…Our next question is what structural features on avidin are responsible for the strong interactions with dopamine or its oxidation products and subsequent oligomeric intermediates. Based on our preliminary experimental results, the modification of glycosyl residues on the surface of avidin with aryl boronic acid groups can only slightly reduce the deceleration effects of avidin on dopamine polymerization . Apart from glycosylation, another significant difference between avidin and streptavidin is the abundant distribution of positively charged amino acids on the surface of avidin.…”

“…11,17,24 In our previous work, we introduced a straightforward method for real-time monitoring of dopamine polymerization kinetics using RhB as a fluorescent indicator. 11,12 This approach takes advantage of pDA's high binding affinity for RhB, which efficiently quenches the fluorescence of RhB. Among the proteins we preliminarily examined, including deoxyribonuclease I (DNase I, M r 31 kDa, pI 4.9, KE 2), bovine serum albumin (BSA, M r 66.5 kDa, pI 4.7, KE 4), trypsin (Try, M r 24 kDa, pI 10.5, KE 0), lysozyme (Lyz, M r 14.5 kDa, pI 11, KE 0), avidin (AVD, M r 68 kDa, pI 10.5, KE 12) and others, only a few proteins (e.g., DNase I and BSA) exhibited a slight acceleration effect on the rapid entrapment and quenching of RhB fluorescence by the resulting pDA.…”

“…Based on our preliminary experimental results, the modification of glycosyl residues on the surface of avidin with aryl boronic acid groups can only slightly reduce the deceleration effects of avidin on dopamine polymerization. 12 Apart from glycosylation, another significant difference between avidin and streptavidin is the abundant distribution of positively charged amino acids on the surface of avidin. Therefore, we attempted to modify the lysine residues on the avidin surface through acetylation or benzoylation to potentially alter their influence on the formation of pDA.…”

“…One remarkable characteristic of dopamine is its tendency to spontaneously undergo autoxidative self-polymerization in alkaline conditions, resulting in the formation of a bioinspired polymer known as polydopamine (pDA) on various surfaces at room temperature. − The unique properties of pDA coatings, coupled with their exceptional biocompatibility and potential for postfunctionalization, have positioned pDA as an increasingly valuable material in a wide range of applications, including energy storage, environmental remediation, cell encapsulation, and drug delivery . Due to the high hydrophilicity, no requirement of additional initiators or cross-linking agents and intrinsic tendency to deposit onto the surface with preimmobilized template protein molecules, dopamine polymerization was further made an attractive reaction for creating molecularly imprinted polymers (MIPs) that exhibit specific recognition capability, multifunctionality, ease of production, and high stability. − …”

“…To monitor the kinetics of dopamine polymerization in the presence of various proteins, we previously developed a straightforward method leveraging the strong binding affinity between pDA and rhodamine B (RhB). , The significant quenching of RhB fluorescence after binding to pDA enabled us to clearly observe how several proteins slowed down the polymerization reaction in real-time by analyzing the fluorescence curves. In this study, we have expanded our investigation to encompass a wider range of protein examples, including three avidin analogues (avidin, streptavidin, and neutravidin) commonly used in the fields of nanotechnology and biotechnology, several representative nucleases involved in DNA repair and recombination, and a typical glycoprotein, erythropoietin (EPO).…”