Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins

Kim, S.,Ko, W.,Sung, B.H.,Kim, S.C.,Lee, H.S. Elsevier/Pergamon 2016 Bioorganic & medicinal chemistry Vol.24 No.22

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance.

Web-Based Computational System for Protein-Protein Interaction Inference

Kim, Ki-Bong Korea Information Processing Society 2012 Journal of information processing systems Vol.8 No.3

Recently, high-throughput technologies such as the two-hybrid system, protein chip, Mass Spectrometry, and the phage display have furnished a lot of data on protein-protein interactions (PPIs), but the data has not been accurate so far and the quantity has also been limited. In this respect, computational techniques for the prediction and validation of PPIs have been developed. However, existing computational methods do not take into account the fact that a PPI is actually originated from the interactions of domains that each protein contains. So, in this work, the information on domain modules of individual proteins has been employed in order to find out the protein interaction relationship. The system developed here, WASPI (Web-based Assistant System for Protein-protein interaction Inference), has been implemented to provide many functional insights into the protein interactions and their domains. To achieve those objectives, several preprocessing steps have been taken. First, the domain module information of interacting proteins was extracted by taking advantage of the InterPro database, which includes protein families, domains, and functional sites. The InterProScan program was used in this preprocess. Second, the homology comparison with the GO (Gene Ontology) and COG (Clusters of Orthologous Groups) with an E-value of $10^{-5}$, $10^{-3}$ respectively, was employed to obtain the information on the function and annotation of each interacting protein of a secondary PPI database in the WASPI. The BLAST program was utilized for the homology comparison.

위암 환자의 위암조직과 위액에서 위암에 특이한 단백에 관한 연구

박경남,신창록,고재경 한양대학교 의과대학 1994 한양의대 학술지 Vol.14 No.1

In order to investigate cancer specific proteins associated with stomach cancer, proteins of stomach cancer tissue and gastric juice from patients with stomach cancer were separated by a DEAE-cellulose column chromatography, high performance liquid chromatography(HPLC) and polyacrylamide gel electrophoresis, and the results were compared with those from control tissue and gastric juice. Protein concentration of cancer tissue and gastric juice of patients with stomach cancer was significantly increased and positive rate of protein contents in stomach cancer tissue and gastric juice as a marker for stomach cancer was high, indicating the possible use of of the protein contents as a biochemical marker for the stomach cancer. Proteins in the stomach cancer tissue were separated by a DEAE cellulose column chromatography into 6 peak proteins, of which a single peak protein (peak Ⅲb) was found to be specific to the cancer and proteins in the gastric juice from patient with stomach cancer were separated into 8 peak proteins, of which three protein peaks(peak Ⅱ, Ⅲ and Ⅳb) were specific to the cancer. Peak Ⅰ and Ⅴ proteins isolated from both cancer tissue and gastric juice from patient with stomach cancer were observed to be activated, suggesting a possible role of these proteins in carcinogenesis and suppression of the stomach cancer. Isolation patterns for proteins of the stomach cancer tissue appeared to be different form those of the control tissue, showing presence of more than 8 protein bands specific to the cancer and disappearance of more than 2 protein bands specific to the cancer and disappearance of more than 2 protein bands from the stomach cancer tissue. The results indicated that changes in proteins of cancer tissue and gastric juice from patient with stomach cancer did not take place in a single protein, but did occur in multiple proteins. Qualitative changes in the proteins were variable in nature, such as presence of cancer specific proteins, disappearance of proteins from the cancer tissue and gastric juice and activiation of the proteins.

New Fast BiFC Plasmid Assay System for <i>in Vivo</i> Protein-Protein Interactions

Kim, Myung-Hwa,Roh, Hee-Eun,Lee, Min-Nyung,Hur, Man-Wook S. Karger AG 2007 CELLULAR PHYSIOLOGY AND BIOCHEMISTRY Vol.20 No.6

<P>In this age of massive genetic and protein information, a fast and reliable method of studying in vivo protein-protein interactions is necessary. We have developed a novel system that can overcome limitations of existing assay methods. This new method adopts two existing systems for fast analysis of diverse protein-protein interactions. For rapid, large-scale cloning, we adopted the Gateway system and developed novel destination vectors containing YFP N-terminus (YN) or YFP C-terminus (YC) to visualize protein-protein interactions <I>in vivo</I> using bimolecular fluorescence complementation (BiFC). Using this system, we investigated molecular interactions among the three POZ-domain regulatory proteins mAPM-1, LRF, KLHL10 that belong to a subgroup of human POZ-domain proteins, and showed that the POZ-domains of mAPM-1, LRF and KLHL10 could form both homodimers and heterodimers. This new method is a highly efficient, sensitive and specific assay method for protein-protein interaction <I>in vivo</I>.</P><P>Copyright © 2007 S. Karger AG, Basel</P>

Essentiality of Hub Proteins in Protein-protein Interaction Networks of Yeast

Jea Woon Ryu,이윤경,강태호,유재수,정진수,박별나,김학용,여명호 한국물리학회 2010 THE JOURNAL OF THE KOREAN PHYSICAL SOCIETY Vol.56 No.5

Scale-free protein interaction networks contain a small number of highly connected proteins, called hubs, and a large number of poorly connected proteins. Recently, several independent studies have elucidated that hub proteins are more likely to be essential to cell function than non-hub proteins. Deletion of a hub protein is more likely to be lethal than deletion of a non-hub protein. This concept defines the centrality-lethality rule; it indicates the importance of hub proteins in a complex protein network and the significance of the network architecture. Determination of the link number for a hub protein is obscure. Therefore, it is important to decide how many link numbers the hub proteins have. Here, we propose a new approach for determining the link number of hub proteins. Hub links were counted by locating the intersection point between the power-law distributions of essential and non-essential proteins. Application of this method to the Uetz database yielded an estimate of seven for the minimum number of hub protein links in yeast. Other public database (Ito, DIP,SGD, and BioGRID) predicted a different number of hub protein links. To assess the reliability of the centrality-lethality rule, we examined the essentiality of hub proteins in the protein interaction networks defined within each of the five public datasets: Uetz, Ito, DIP, SGD, and BioGRID. All five sites indicated that hub proteins were more likely to be essential than were non-hub proteins. This new method for determining the number of hub links is a useful tool for hub proteins.

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>

Mining Proteins Associated with Oral Squamous Cell Carcinoma in Complex Networks

Liu, Ying,Liu, Chuan-Xia,Wu, Zhong-Ting,Ge, Lin,Zhou, Hong-Mei Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.8

The purpose of this study was to construct a protein-protein interaction (PPI) network related to oral squamous cell carcinoma (OSCC). Each protein was ranked and those most associated with OSCC were mined within the network. First, OSCC-related genes were retrieved from the Online Mendelian Inheritance in Man (OMIM) database. Then they were mapped to their protein identifiers and a seed set of proteins was built. The seed proteins were expanded using the nearest neighbor expansion method to construct a PPI network through the Online Predicated Human Interaction Database (OPHID). The network was verified to be statistically significant, the score of each protein was evaluated by algorithm, then the OSCC-related proteins were ranked. 38 OSCC related seed proteins were expanded to 750 protein pairs. A protein-protein interaction nerwork was then constructed and the 30 top-ranked proteins listed. The four highest-scoring seed proteins were SMAD4, CTNNB1, HRAS, NOTCH1, and four non-seed proteins P53, EP300, SMAD3, SRC were mined using the nearest neighbor expansion method. The methods shown here may facilitate the discovery of important OSCC proteins and guide medical researchers in further pertinent studies.

단백질 어레이의 발달과 응용 그리고 전망

최우봉 동의대학교 산업기술개발연구소 2005 産業技術硏究誌 Vol.19 No.-

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of proteinchips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.

Effective non-denaturing purification method for improving the solubility of recombinant actin-binding proteins produced by bacterial expression

Chung, Jeong Min,Lee, Sangmin,Jung, Hyun Suk Elsevier 2017 Protein expression and purification Vol.133 No.-

<P><B>Abstract</B></P> <P>Bacterial expression is commonly used to produce recombinant and truncated mutant eukaryotic proteins. However, heterologous protein expression may render synthesized proteins insoluble. The conventional method used to express a poorly soluble protein, which involves denaturation and refolding, is time-consuming and inefficient. There are several non-denaturing approaches that can increase the solubility of recombinant proteins that include using different bacterial cell strains, altering the time of induction, lowering the incubation temperature, and employing different detergents for purification. In this study, we compared several non-denaturing protocols to express and purify two insoluble 34 kDa actin-bundling protein mutants. The solubility of the mutant proteins was not affected by any of the approaches except for treatment with the detergent sarkosyl. These results indicate that sarkosyl can effectively improve the solubility of insoluble proteins during bacterial expression.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The expression and purification condition for truncated actin binding proteins was optimized from <I>Escherichia coli</I> strain. </LI> <LI> The treatment of sarkosyl-detergent increases the solubility of bacterial recombinant proteins. </LI> <LI> Minimizing concentration of sarkosyl was suggested for enhancing the production of soluble proteins. </LI> </UL> </P>

Case Report : A Case of Protein Supplement Effect in Protein-Losing Enteropathy

( Hyun Jeong Lee ),( Mi Yong Rha ),( Young Yun Cho ),( Eun Ran Kim ),( Dong Kyung Chang ) 한국임상영양학회 2012 Clinical Nutrition Research Vol.1 No.1

The objective of this article is to report improvement of nutritional status by protein supplements in the patient with proteinlosing enteropathy. The patient was a female whose age was 25 and underwent medical treatment of Crohn``s disease, an in- flammatory bowl disease, after diagnosis of cryptogenic multifocal ulcerous enteritis. The weight was 33.3 kg (68% of IBW) in the severe underweight and suffered from ascites and subcutaneous edema with hypoalbuminemia (1.3 g/dL) at the time of hospitalization. The patient consumed food restrictively due to abdominal discomfort. Despite various attempts of oral feeding, the levels of calorie and protein intake fell into 40-50% of the required amount, which was 800-900 kcal/d (24-27 kcal/ kg/d) for calorie and 34 g/d (1 g/kg/d) for protein. It was planned to supplement the patient with caloric supplementation (40- 50 kcal/kg) and protein supplementation (2.5 g/kg) to increase body weight and improve hypoproteinemia. It was also planned to increase the level of protein intake slowly to target 55 g/d in about 2 weeks starting from 10 g/d and monitored kidney load with high protein supplementation. The weight loss was 1.0 kg when the patient was discharged from the hospital (hospitalization periods of 4 weeks), however, serum albumin was improved from 1.3 g/dL to 2.5 g/dL and there was no abdominal discomfort. She kept supplement of protein at 55 g/d for 5 months after the discharge from the hospital and kept it at 35 g/d for about 2 months and then 25 g/d. The body weight increased gradually from 32.3 kg (65% of IBW) to 44.0 kg (89% of IBW) by 36% for the period of F/u and serum albumin was kept above 2.8 g/dL without intravenous injection of albumin. The performance status was improved from 4 points of ``very tired`` to 2 points of ``a little tired`` out of 5-point scale measurement and the use of diuretic stopped from the time of 4th month after the discharge from the hospital owing to improvement in edema and ascites. During this period, the results of blood test such as BUN, Cr, and electrolytes were within the normal range. In conclusion, hypoproteinemia and weight loss were improved by increasing protein intake through utilization of protein supplements in proteinlosing enteropathy.