Virus-based surface patterning of biological molecules, probes, and inorganic materials

Ahn, S.,Jeon, S.,Kwak, E.A.,Kim, J.M.,Jaworski, J. Elsevier 2014 Colloids and Surfaces B Vol.122 No.-

An essential requirement for continued technological advancement in many areas of biology, physics, chemistry, and materials science is the growing need to generate custom patterned materials. Building from recent achievements in the site-specific modification of virus for covalent surface tethering, we show in this work that stable 2D virus patterns can be generated in custom geometries over large area glass surfaces to yield templates of biological, biochemical, and inorganic materials in high density. As a nanomaterial building block, filamentous viruses have been extensively used in recent years to produce materials with interesting properties, owing to their ease of genetic and chemical modification. By utilizing un-natural amino acids generated at specific locations on the filamentous fd bacteriophage protein coat, surface immobilization is carried out on APTES patterned glass resulting in precise geometries of covalently linked virus material. This technique facilitated the surface display of a high density of virus that were labeled with biomolecules, fluorescent probes, and gold nanoparticles, thereby opening the possibility of integrating virus as functional components for surface engineering.

Covalent crowding strategy for trypsin confined in accessible mesopores with enhanced catalytic property and stability

Cheng Zhou,Shubao Shen,Bo Jiang,Zecui Sheng,Shemin Zhu 한국화학공학회 2011 Korean Journal of Chemical Engineering Vol.28 No.3

Chemically modified macromolecules were assembled with adsorptive trypsin in mesoporous silica foams (MCFs) to establish covalent linkage. Effects of catalytic properties and stability of immobilized trypsin were examined. The addition of chemically modified protein (BSA) and polysaccharide (ficoll) to the immobilized trypsin exhibited high coupled yield (above 90%) and relative activities (174.5% and 175.9%, respectively), showing no protein leaching after incubating for 10 h in buffers. They showed broader pH and temperature profiles, while the half life of thermal stability of BSA-modified preparation at 50 ℃ increased to 1.3 and 2.3 times of unmodified and free trypsin,respectively. The modified trypsin in aqueous-organic solvents exhibited 100% activity after 6 h at 50 ℃. The kinetic parameters of trypsin preparations and suitable pore diameter of MCFs warranted compatibility of covalent modification for substrate transmission. The covalent crowding modification for immobilized trypsin in nanopores establishes suitable and accessible microenvironment and renders possibility of biological application.

Adaptive Responses to Electrophilic Stress and Reactive Sulfur Species as their Regulator Molecules

Yoshito Kumagai,Masahiro Akiyama,Takamitsu Unoki 한국독성학회 2019 Toxicological Research Vol.35 No.4

We are exposed to numerous xenobiotic electrophiles on a daily basis through the environment, lifestyle, and dietary habits. Although such reactive species have been associated with detrimental effects, recent accumulated evidence indicates that xenobiotic electrophiles appear to act as signaling molecules. In this review, we introduce our findings on 1) activation of various redox signaling pathways involved in cell proliferation, detoxification/ excretion of electrophiles, quality control of cellular proteins, and cell survival during exposure to xenobiotic electrophiles at low concentrations through covalent modification of thiol groups in sensor proteins, and 2) negative regulation of reactive sulfur species (RSS) in the modulation of redox signaling and toxicity caused by xenobiotic electrophiles.

Adaptive Responses to Electrophilic Stress and Reactive Sulfur Species as their Regulator Molecules

Kumagai, Yoshito,Akiyama, Masahiro,Unoki, Takamitsu Korean Society of ToxicologyKorea Environmental Mu 2019 Toxicological Research Vol.35 No.4

We are exposed to numerous xenobiotic electrophiles on a daily basis through the environment, lifestyle, and dietary habits. Although such reactive species have been associated with detrimental effects, recent accumulated evidence indicates that xenobiotic electrophiles appear to act as signaling molecules. In this review, we introduce our findings on 1) activation of various redox signaling pathways involved in cell proliferation, detoxification/excretion of electrophiles, quality control of cellular proteins, and cell survival during exposure to xenobiotic electrophiles at low concentrations through covalent modification of thiol groups in sensor proteins, and 2) negative regulation of reactive sulfur species (RSS) in the modulation of redox signaling and toxicity caused by xenobiotic electrophiles.

Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins

Cho, H.,Jaworski, J. Elsevier 2014 Colloids and Surfaces B Vol.122 No.-

The covalent immobilization of proteins onto surfaces is an essential aspect of several fields of research, including proteomics, sensing, heterogeneous biocatalysis, and more broadly biotechnology. Site-specific, covalent attachment of proteins has been achieved in recent years by the use of expanded genetic codes to produce proteins with controlled placement of un-natural amino acids bearing bio-orthogonal functional groups. Unfortunately, the complexity of developing such systems is impractical for most laboratories; hence, a less complicated approach to generating un-natural amino acid side-chains has been employed. Utilizing a straightforward reaction with formylglycine generating enzyme, we use the site-specific modification of engineered proteins to yield un-natural amino acid side-chains for protein immobilization. Using this approach, we demonstrate the controlled immobilization of various enzymes onto a variety of amine coated surfaces. Our results reveal reusability of the immobilized enzymes via this strategy, and furthermore, we find the activity of the immobilized enzymes to remain even after a month of use indicating significant stability of the linkage.

Post-immobilization of Modified Macromolecular Reagents using Assembled Penicillin Acylase for Microenvironmental Regulation of Nanopores and Enhancement of Enzyme Stability

Cheng Zhou,Shemin Zhu,Xiuming Wu,Bo Jiang,Tao Cen,Shubao Shen 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.3

Penicillin acylase (PA) is known to regulate the microenvironment of nanospores. In this study, nanopores containing chemically-modified macromolecules co-assembled with immobilized PA were constructed. We also investigated the various types of functionalized mesocellular siliceous foams (MCFs) commonly used for the immobilization of PA by measuring the catalytic performance and stability of each PA preparation. Amino-MCF activated by p-benzoquinone was chosen as the optimum support for PA immobilization. Successful modification of macromolecules was verified by FT-IR and ultraviolet (UV)spectroscopy. The specific activity of PA co-assembled with dextran 10 k was 99.1 U/mg, which was 1.5-fold that of pristine immobilized PA, while the optimum pH was shifted to neutral. Compared to pristine immobilized and free PA, the optimum temperatures for the modified PA were 5 and 10oC higher, respectively. The residual activity of the ficoll derivative of PA after treatment at 50oC for 6 h was 70%, and this was later increased to 214.5% compared to that of pristine immobilized PA. The dextran 10 k derivative of PA exhibited 90.2% residual activity after 25times of continuous use. The results show that chemicallymodified macromolecules co-assembled with PA in amino-MCF provided a suitable microenvironment for enzyme stability.

Identification of Novel Irreversible Inhibitors of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Haemophilus influenzae

( Seong Gu Han ),( Won Kyu Lee ),( Bong Suk Jin ),( Ki In Lee ),( Hyeong Ho Lee ),( Yeon Gyu Yu ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.3

Uridinediphospho-N-acetylglucosamine enolpyruvyl transferase (MurA, E.C. 2.5.1.7) is an essential bacterial enzyme that catalyzes the first step of the cell wall biosynthetic pathway, which involves the transfer of an enolpyruvyl group from phosphoenolpyruvate to uridinediphospho-Nacetylglucosamine. In this study, novel inhibitors of Haemophilus influenzae MurA (Hi MurA) were identified using high-throughput screening of a chemical library from the Korea Chemical Bank. The identified compounds contain a quinoline moiety and have much lower effective inhibitory concentrations (IC50) than fosfomycin, a wellknown inhibitor of MurA. These inhibitors appear to covalently modify the sulfhydryl group of the active site cysteine (C117), since the C117D mutant Hi MurA was not inhibited by these compounds and excess dithiothreitol abolished their inhibitory activities. The increased mass value of Hi MurA after treatment with the identified inhibitor further confirmed that the active-site cysteine residue of Hi MurA is covalently modified by the inhibitor.