Molecular Bioprocessing as an Emerging Paradigm for Advancing Medicine

Jonathan S. DORDICK 한국생물공학회 2016 한국생물공학회 학술대회 Vol.2016 No.4

Exploiting the Interface of Biomolecular Engineering and Materials Science to Generate New Therapeutic Outcomes

Jonathan S. DORDICK 한국생물공학회 2017 한국생물공학회 학술대회 Vol.2017 No.4

학술발표회 초록 - 특강 ( 생물화공 (Ⅲ) )

류근갑(Keun Garp Ryu),김중배(Joong Bae Kim),J . S . Dordick(Jonathan S . Dordick) 한국화학공학회 1993 추계학술발표논문집 Vol.- No.-

Review : Cell-Based Assay Design for High-Content Screening of Drug Candidates

( Gregory Nierode ),( Paul S Kwon ),( Jonathan S Dordick ),( Seok Joon Kwon ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.2

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

Polymers from Biocatalysts

YOO, YOUNG JE,Joo, Hyun,Dordick, Jonathan S . 한국화학공학회 1998 Korean Journal of Chemical Engineering Vol.15 No.4

Recenfly, there has been great interest m enzyme-catalyzed polymer synthesis, particularly as an alernative to conventional polymer chemistry. Highly specific and stable biocatalysts can result in increased yields and reduced capital requirements and production costs. Moreover, the use of biocatalysts can minimize the formation of unwanted byproducts and thus reduce separation cost. Enzymatic synthesis of polymer is especially preferred when molecular regularity such as stereoselectivity or regiospecificity is required. In this article, the use of enzymes in polymer.synthesis is drscussed together with potential applications.

Flexible Peptide Linkers Enhance the Antimicrobial Activity of Surface-Immobilized Bacteriolytic Enzymes

Wu, Xia,Fraser, Keith,Zha, Jian,Dordick, Jonathan S. American Chemical Society 2018 ACS APPLIED MATERIALS & INTERFACES Vol.10 No.43

<P>Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity, whereas peptide linkers, although ubiquitous in protein engineering, are much less explored in enzyme immobilization. Here, we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti-<I>Staphylococcus aureus</I> surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (His-tag) fused at the C-terminus of Lst, respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag, the immobilized enzymes killed >99.5% of <I>S. aureus</I> ATCC 6538 cells (10<SUP>8</SUP> CFU/mL) within 3 h in buffer and could be reused multiple times without significant loss of activity. In contrast, immobilized Lst without a peptide linker was less active/stable. Molecular modeling of Lst-linker-affinity tag constructs illustrated that the presence of the peptide linkers enhanced the molecular flexibility of the proximal Lst binding domain, which interacts with the bacterial substrate, and such increased flexibility correlated with increased antimicrobial activity. We further show that Lst immobilized onto NiNTA beads retained the ability to kill ∼99% of a 10<SUP>8</SUP> CFU/mL microbial challenge even in the presence of 1% of a commercial anionic surfactant, C12-14 alcohol EO 3:1 sodium sulfate, when the Lst construct contained a decapeptide linker containing glycine, serine, and alanine residues. This linker-assisted immobilization strategy could be extended to an unrelated lytic enzyme, the endolysin PlyPH, to target <I>Bacillus anthracis</I> Sterne cells either in buffer or in the presence of anionic surfactants. Our approach, therefore, provides a facile route to the use of antimicrobial enzymes on surfaces.</P> [FIG OMISSION]</BR>

Unprotonated Short-Chain Alkylamines Inhibit Staphylolytic Activity of Lysostaphin in a Wall Teichoic Acid-Dependent Manner

Wu, Xia,Kwon, Seok Joon,Kim, Domyoung,Zha, Jian,Mora-Pale, Mauricio,Dordick, Jonathan S. American Society for Microbiology 2018 Applied and environmental microbiology Vol.84 No.14

<P>Lysostaphin (Lst) is a potent bacteriolytic enzyme that kills <I>Staphylococcus aureus</I>, a common bacterial pathogen of humans and animals. With high activity against both planktonic cells and biofilms, Lst has the potential to be used in industrial products, such as commercial cleansers, for decontamination. However, Lst is inhibited in the presence of monoethanolamine (MEA), a chemical widely used in cleaning solutions and pharmaceuticals, and the underlying mechanism of inhibition remains unknown. In this study, we examined the cell binding and killing capabilities of Lst against <I>S. aureus</I> ATCC 6538 in buffered salt solution with MEA at different pH values (7.5 to 10.5) and discovered that only the unprotonated form of MEA inhibited Lst binding to the cell surface, leading to low Lst activity, despite retention of its secondary structure. This reduced enzyme activity could be largely recovered via a reduction in wall teichoic acid (WTA) biosynthesis through tunicamycin treatment, indicating that the suppression of Lst activity was dependent on the presence and amount of WTA. We propose that the decreased cell binding and killing capabilities of Lst are associated with the influence of uncharged MEA on the conformation of WTA. A similar effect was confirmed with other short-chain alkylamines. This study offers new insight into the impact of short-chain alkylamines on both Lst and WTA structure and function and provides guidance for the application of Lst in harsh environments.</P><P><B>IMPORTANCE</B> Lysostaphin (Lst) effectively and selectively kills <I>Staphylococcus aureus</I>, the bacterial culprit of many hospital- and community-acquired skin and respiratory infections and food poisoning. Lst has been investigated in animal models and clinical trials, industrial formulations, and environmental settings. Here, we studied the mechanistic basis of the inhibitory effect of alkylamines, such as monoethanolamine (MEA), a widely used chemical in commercial detergents, on Lst activity, for the potential incorporation of Lst in disinfectant solutions. We have found that protonated MEA has little influence on Lst activity, while unprotonated MEA prevents Lst from binding to <I>S. aureus</I> cells and hence dramatically decreases the enzyme's bacteriolytic efficacy. Following partial removal of the wall teichoic acid, an important component of the bacterial cell envelope, the inhibitory effect of unprotonated MEA on Lst is reduced. This phenomenon can be extended to other short-chain alkylamines. This mechanistic report of the impact of alkylamines on Lst functionality will help guide future applications of Lst in disinfection and decontamination of health-related commercial products.</P>

Immobilization of Glucose Oxidase on Graphene Oxide for Highly Sensitive Biosensors

Hong, Sung-Gil,Kim, Jae Hyun,Kim, Ryang Eun,Kwon, Seok-Joon,Kim, Dae Woo,Jung, Hee-Tae,Dordick, Jonathan S.,Kim, Jungbae Korean Society for Biotechnology and Bioengineerin 2016 Biotechnology and Bioprocess Engineering Vol.21 No.4

Glucose oxidase (GOx) was immobilized onto graphene oxide (GRO) via three different preparation methods: enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC), and enzyme adsorption, precipitation and crosslinking (EAPC). EAPC formulations, prepared via enzyme precipitation with 60% ammonium sulfate, showed 1,980 and 1,630 times higher activity per weight of GRO than those of EA and EAC formulations, respectively. After 59 days at room temperature, EAPC maintained 88% of initial activity, while EA and EAC retained 42 and 45% of their initial activities, respectively. These results indicate that the steps of precipitation and crosslinking in the EAPC formulation are critical to achieve high enzyme loading and stability of EAPC. EA, EAC and EAPC were used to prepare enzyme electrodes for use as glucose biosensors. Optimized EAPC electrode showed 93- and 25-fold higher sensitivity than EA and EAC, respectively. To further increase the sensitivity of EAPC electrode, multi-walled carbon nanotubes (MWCNTs) were mixed with EAPC for the preparation of enzyme electrode. Surprisingly, the EAPC electrode with additional 99.5 wt% MWCNTs showed 7,800-fold higher sensitivity than the EAPC electrode without MWCNT addition. Immobilization and stabilization of enzymes on GRO via the EAPC approach can be used for the development of highly sensitive biosensors as well as to achieve high enzyme loading and stability.

Unnatural Polyketide Analogues Selectively Target the HER Signaling Pathway in Human Breast Cancer Cells

Kwon, Seok Joon,Kim, Moon Il,Ku, Bosung,Coulombel, Lydie,Kim, Jin-Hwan,Shawky, Joseph H.,Linhardt, Robert J.,Dordick, Jonathan S. WILEY-VCH Verlag 2010 Chembiochem Vol.11 No.4

<P>Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets. Several plant polyphenols including the type III polyketide synthase products (genistein, curcumin, resveratrol, and epigallocatechin-3-galate) possess chemopreventive activity, primarily as a result of RTK inhibition. However, only a small fraction of the polyphenolic structural universe has been evaluated. Along these lines, we have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogues with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme A (CoA) and methylmalonyl-CoA as extender substrates. The resulting polyketide analogues possessed a similar structural polyketide backbone (aromatic-2-pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT-474 cells resulted in identification of several trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of nontransformed MCF-10A breast cells (IC<SUB>50</SUB>>100 μM). Specifically, 4-trifluoromethylcinnamoyl pyrone (compound 2 e) was highly potent (IC<SUB>50</SUB><200 nM) among the test compounds toward proliferation of several breast cancer cell lines. This breadth of activity likely stems from the ability of compound 2 e to inhibit the phosphorylation of HER1, HER2, and HER3. Therefore, these polyketide analogues might prove to be useful drug candidates for potential breast cancer therapy.</P> <B>Graphic Abstract</B> <P>Multiple HER TK inhibitor: In vitro precursor-directed syntheses of polyketide analogues resulted in the identification of several “unnatural” natural products that suppressed the HER-PI3K-Akt signaling pathway, as well as having multiple HER TK inhibitory activity. <img src='wiley_img_2010/14394227-2010-11-4-CBIC200900674-content.gif' alt='wiley_img_2010/14394227-2010-11-4-CBIC200900674-content'> </P>

Selective Killing of Pathogenic Bacteria by Antimicrobial Silver Nanoparticle-Cell Wall Binding Domain Conjugates

Kim, Domyoung,Kwon, Seok-Joon,Wu, Xia,Sauve, Jessica,Lee, Inseon,Nam, Jahyun,Kim, Jungbae,Dordick, Jonathan S. American Chemical Society 2018 ACS APPLIED MATERIALS & INTERFACES Vol.10 No.16

<P>Broad-spectrum antibiotics indiscriminately kill bacteria, removing nonpathogenic microorganisms and leading to evolution of antibiotic resistant strains. Specific antimicrobials that could selectively kill pathogenic bacteria without targeting other bacteria in the natural microbial community or microbiome may be able to address this concern. In this work, we demonstrate that silver nanoparticles, suitably conjugated to a selective cell wall binding domain (CBD), can efficiently target and selectively kill bacteria. As a relevant example, CBD<SUP>BA</SUP> from <I>Bacillus anthracis</I> selectively bound to <I>B. anthracis</I> in a mixture with <I>Bacillus subtilis</I>, as well in a mixture with <I>Staphylococcus aureus</I>. This new biologically-assisted hybrid strategy, therefore, has the potential to provide selective decontamination of pathogenic bacteria with minimal impact on normal microflora.</P> [FIG OMISSION]</BR>