Biomolecular engineering for nanobio/bionanotechnology

Nagamune Teruyuki 나노기술연구협의회 2017 Nano Convergence Vol.4 No.9

Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.

Engineering Thermal Properties of Elastin-like Polypeptides by Incorporation of Unnatural Amino Acids in a Cell-free Protein Synthesis System

Christy Catherine,오수진,이경호,민승의,원종인,윤형돈,김동명 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3

Based on the central dogma of protein synthesis, traditional methods for protein engineering require that altering protein structure and function must be accompanied by changing the nucleotide sequence of the genes encoding the protein. However, the preparation of a template gene for each individual protein requires a great deal of time and effort, thereby limiting the throughput and scope of studying engineered proteins. In this study, we describe translation-level engineering of proteins using cell-free protein synthesis. Taking advantage of the promiscuity of aminoacyl tRNA synthetases in accepting structurally similar amino acid analogues, unnatural amino acids were introduced into elastin-like polypeptides in place of the corresponding cognate amino acids. Through the incorporation of various analogues and starting from the same gene, the phase transition temperatures of elastin-like polypeptides became tunable. Our results demonstrate the usefulness of cell-free protein synthesis for protein engineering using unnatural amino acids without the need for cloning.

Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

Suh, Jin Sook,Lee, Jue Yeon,Choi, Yoon Jung,You, Hyung Keun,Hong, Seong-Doo,Chung, Chong Pyoung,Park, Yoon Jeong Dove Medical Press 2014 INTERNATIONAL JOURNAL OF NANOMEDICINE Vol.9 No.-

<P>Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue-engineering purposes.</P>

Intein-mediated Protein Engineering for Biosensor Fabrication

전현진,이민형,장원희,권영은 한국바이오칩학회 2016 BioChip Journal Vol.10 No.4

Intein-mediated protein engineering has become a valuable tool for biosensing applications. Expressed protein ligation and protein trans-splicing were used to generate proteins with necessary tags or cyclic peptides that can be used to build various biosensing platforms such as biochips or sensor proteins. Especially the use of split-inteins that carry out conditional protein splicing reaction enabled the fabrication of various genetically-encoded biosensors that can monitor various signaling events in vivo including protein- protein interactions and protein translocalization. Biological targets activated split-inteins to generate functioning reporter molecules, such as luciferase and autofluorescent proteins. We here review various sensing platforms that utilize intein-mediated protein engineering technology mainly focusing on cell-based biosensors.

Enhanced production of unnatural amino acid-containing proteins in a cell-free protein synthesis system

Lee, K.H.,Catherine, C.,Kim, D.M. Korean Society of Industrial and Engineering Chemi 2016 Journal of industrial and engineering chemistry Vol.37 No.-

<P>Replacement of canonical amino acids with unnatural amino acids (UAAs) can provide proteins with novel physicochemical properties and biological functions. In this study, as an alternative option to conventional cell-based methods, we used a cell-free protein synthesis system as a flexible platform for facile and efficient production of UAA-containing proteins. We designed a cell-free protein synthesis system derived from the extract of Escherichia coli cells to maximize the selective incorporation of UAAs into the protein structure. First, for the purpose of avoiding competitive incorporation of canonical amino acids and UAAs, the cell extract was extensively washed using a diafiltration process to remove residual amino acids, thereby making the protein synthesis reaction completely dependent upon the exogenous addition of amino acids. In addition, the relatively low affinity of UAAs for cognate aminoacyl-tRNA synthetase was kinetically overcome by increasing the concentration of UAAs to nonphysiological levels. As a result of these modifications of the cell-free synthesis systems, we were able to produce UAA-containing proteins at comparable yields to those of proteins made of canonical amino acids. (C) 2016 Published by Elsevier B.V. on behalf of The Korean Society of Industrial and Engineering Chemistry.</P>

엘리트 레슬링 선수들과 이공계 대학생들간의 모발을 이용한 유전정보 전달체계의 D.N.A. R.N.A. 및 Protein 비교연구

김태우 ( Tae Woo Kim ) 한국스포츠리서치 2007 한국 스포츠 리서치 Vol.18 No.4

The purpose of this study was to compare the gene and protein on sportsman and college man. In edition of highly trained young male athletes was investigated. Ten subjects volunteered (a state amateur wrestling) and age matched control(N=10). Measurements of DNA, RNA, Protein(ug/protein) utilizing Carbonyl contents determined using 2,4-dinitrophenylhydrazine in proteins solubilized hair. AGPC(acid guanidine-phenol-chloform) method used obtained for RNA in hair. ph8.0, 0.1 mol/l NaCl, 5 mmol/l EDTA), TNE buffer(10mmol/l Tris-Cl cooling method were obtained for DNA. in hair. An addition to changes in hair DNA, total RNA, Protein were observed by SDS-PAG electrophretical analysis. Results of this study are following. 1. Protein % is 16.9% in control group, 21.6% in wrestler group, wrestler group has a significantly more a amount of Protein % than control group(p<.01). 2. It is significantly different in amounts of Total RNA(ug/protein) between groups(p<.05). 3. It is significantly different in amounts of DNA(ug/protein) between groups(p<.05). Conclusionally, this study indicate that improvement of ``Creative Evolution`` in RNA, DNA, and Protein genes changed by exercise or sports. Moreover exercise was effect on Central Dogma with DNA makes RNA makes Protein. This study is expected to contribute the area of sports science, medicine, hereafter more effort is required to establish the relation between gene(informational macromolecule) alters and exercise amount.

In-depth proteomic analysis of <i>Glycine max</i> seeds during controlled deterioration treatment reveals a shift in seed metabolism

Min, Cheol Woo,Lee, Seo Hyun,Cheon, Ye Eun,Han, Won Young,Ko, Jong Min,Kang, Hang Won,Kim, Yong Chul,Agrawal, Ganesh Kumar,Rakwal, Randeep,Gupta, Ravi,Kim, Sun Tae Elsevier 2017 Journal of proteomics Vol.169 No.-

<P><B>Abstract</B></P> <P>Seed aging is one of the major events, affecting the overall quality of agricultural seeds. To analyze the effect of seed aging, soybean seeds were exposed to controlled deterioration treatment (CDT) for 3 and 7days, followed by their physiological, biochemical, and proteomic analyses. Seed proteins were subjected to protamine sulfate precipitation for the enrichment of low-abundance proteins and utilized for proteome analysis. A total of 14 differential proteins were identified on 2-DE, whereas label-free quantification resulted in the identification of 1626 non-redundant proteins. Of these identified proteins, 146 showed significant changes in protein abundance, where 5 and 141 had increased and decreased abundances, respectively while 352 proteins were completely degraded during CDT. Gene ontology and KEGG analyses suggested the association of differential proteins with primary metabolism, ROS detoxification, translation elongation and initiation, protein folding, and proteolysis, where most, if not all, had decreased abundance during CDT. Western blotting confirmed reduced level of antioxidant enzymes (DHAR, APx1, MDAR, and SOD) upon CDT. This in-depth integrated study reveals a major downshift in seed metabolism upon CDT. Reported data here serve as a resource for its exploitation to metabolic engineering of seeds for multiple purposes, including increased seed viability, vigor, and quality.</P> <P><B>Biological significance</B></P> <P>Controlled deterioration treatment (CDT) is one of the major events that negatively affects the quality and nutrient composition of agricultural seeds. However, the molecular mechanism of CDT is largely unknown. A combination of gel-based and gel-free proteomic approach was utilized to investigate the effects of CDT in soybean seeds. Moreover, we utilized protamine sulfate precipitation method for enrichment of low-abundance proteins, which are generally masked due to the presence of high-abundance seed storage proteins. Reported data here serve as resource for its exploitation to metabolic engineering of seeds for multiple purposes, including increased seed viability, vigor, and quality.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Protamine sulfate precipitation was used for enrichment of low-abundance proteins. </LI> <LI> 2DE & label-free quantitative proteomic approach were used to analyze effect of CDT. </LI> <LI> Using this approach, a total of 1640 (14+1626) proteins were identified. </LI> <LI> These proteins were mainly related to the primary metabolism and ROS detoxification. </LI> <LI> Western blotting confirmed reduced level of DHAR, APx1, MDAR, and SOD upon CDT. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Using a Two Species Competitive Binding Model to Predict Expanded Bed Breakthrough of a Recombinant Protein Expressed in a High Cell Density Fermentation

William Kelly,Guy Kamguia,Peter Mullen,Antonio Ubiera,Kent Göklen,Zuyi Huang,Gerard Jones 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.3

Expanded Bed experiments were conducted using a mixed mode (MM) resin to capture and purify a recombinant protein produced in yeast fermentation. Expanded bed breakthrough profiles show an overshoot in column effluent concentration of the target protein in the presence of cells and other broth proteins, similar to that seen by other researchers when loading two competing species onto packed beds. In this research, a numerical model assuming negligible axial dispersion is developed and first validated for columns loads that contain only the target protein. This model is solved by finite differences in a unique way that uses an embedded analytical-solution to increase solution speed and stability. To model expanded bed breakthrough of the target protein in the actual cell broth, it was assumed that the other non-product proteins in the broth compete for MM resin binding sites and might be represented as a second “average” species via a traditional two-component competitive Langmuir isotherm. Estimates of the Langmuir constant and broth concentration of this second species were then calculated from batch adsorption data. Using these parameters for the second species, and other batch-derived parameters for the target protein with this resin, this unique numerical modeling approach provided results that compare favorably to experimental breakthrough data at various flow rates. Finally, the model was employed for a parameter sensitivity analysis that shows which process variables are most important in determining breakthrough time and the shape and magnitude of the concentration overshoot.

Engineering the Cellular Protein Secretory Pathway for Enhancement of Recombinant Tissue Plasminogen Activator Expression in Chinese Hamster Ovary Cells: Effects of CERT and XBP1s Genes

( Azam Rahimpour ),( Behrouz Vaziri ),( Reza Moazzami ),( Leila Nematollahi ),( Farzaneh Barkhordari ),( Leila Kokabee ),( Ahmad Adeli ),( Fereidoun Mahboudi ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.8

Cell line development is the most critical and also the most time-consuming step in the production of recombinant therapeutic proteins. In this regard, a variety of vector and cell engineering strategies have been developed for generating high-producing mammalian cells; however, the cell line engineering approach seems to show various results on different recombinant protein producer cells. In order to improve the secretory capacity of a recombinant tissue plasminogen activator (t-PA)-producing Chinese hamster ovary (CHO) cell line, we developed cell line engineering approaches based on the ceramide transfer protein (CERT) and X-box binding protein 1 (XBP1) genes. For this purpose, CERT S132A, a mutant form of CERT that is resistant to phosphorylation, and XBP1s were overexpressed in a recombinant t-PA-producing CHO cell line. Overexpression of CERT S132A increased the specific productivity of t-PA-producing CHO cells up to 35%. In contrast, the heterologous expression of XBP1s did not affect the t-PA expression rate. Our results suggest that CERTS132A- based secretion engineering could be an effective strategy for enhancing recombinant t- PA production in CHO cells.

Anti-apoptosis Engineering

Kim, Eun-Jeong,Park, Tai-Hyun The Korean Society for Biotechnology and Bioengine 2003 Biotechnology and Bioprocess Engineering Vol.8 No.2

An increased understanding of apoptosis makes anti-apoptosis engineering possible, which is an approach used to inhibit apoptosis for the purpose of therapeutic, or industrial applications in the treatment of the diseases associated with increased apoptosis, or to improve the productivity of animal cell cultures, respectively. Some known anti-apoptosis proteins are the Bcl-2 family, IAP (inhibitor of apoptosis) and Hsps (heat shock proteins), with which anti-apoptosis engineering has progressed. This article reviews anti-apoptosis engineering using known anti-apoptosis compounds, and introduces a 30 K protein, isolated from silkworm hemolymph, as a novel anti-apoptotic protein, that Shows no homology with other known anti-apoptotic proteins. The regulation of apoptosis, using anti-apoptotic proteins and genes originating from the silkworm, Bombyx mori, may provide a new strategy in this field.