Pub Date : 2018-07-18DOI: 10.5772/INTECHOPEN.77337
M. Ijaz, M. Ansari, M. Iqbal
Protein-protein interactions (PPIs) control variety of biological phenomena including development, cell to cell interactions and metabolic processes [1]. The PPIs can be classified into different groups, depending upon their functional and structural properties [2]. Depending upon their persistence, (1) they may be termed as permanent or transient, as characterized by their interaction surface, (2) they may be considered as heterooligomeric or homooligomeric based on their stability, and (3) they may be called as obligate or nonobligate [3]. A blend of these three pairs may develop a protein-protein interaction. For example, a permanent interaction of the protein may be able to form a stable protein complex while on the other hand a transient interaction among the proteins may form a signaling pathway [4].
{"title":"Introductory Chapter: Protein-Protein Interactions and Assays","authors":"M. Ijaz, M. Ansari, M. Iqbal","doi":"10.5772/INTECHOPEN.77337","DOIUrl":"https://doi.org/10.5772/INTECHOPEN.77337","url":null,"abstract":"Protein-protein interactions (PPIs) control variety of biological phenomena including development, cell to cell interactions and metabolic processes [1]. The PPIs can be classified into different groups, depending upon their functional and structural properties [2]. Depending upon their persistence, (1) they may be termed as permanent or transient, as characterized by their interaction surface, (2) they may be considered as heterooligomeric or homooligomeric based on their stability, and (3) they may be called as obligate or nonobligate [3]. A blend of these three pairs may develop a protein-protein interaction. For example, a permanent interaction of the protein may be able to form a stable protein complex while on the other hand a transient interaction among the proteins may form a signaling pathway [4].","PeriodicalId":273381,"journal":{"name":"Protein-Protein Interaction Assays","volume":"32 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"131593993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-07-18DOI: 10.5772/INTECHOPEN.74733
N. Migliaccio, Gennaro Sanità, I. Ruggiero, N. Martucci, C. Sanges, E. Rippa, V. Quagliariello, F. Papale, P. Arcari, A. Lamberti
Besides its canonical role in protein synthesis, the eukaryotic translation elongation fac- tor 1A (eEF1A) is also involved in many other cellular processes such as cell survival and apoptosis. We showed that eEF1A phosphorylation by C-Raf in vitro occurred only in the presence of eEF1A1 and eEF1A2, thus suggesting that both isoforms interacted in cancer cells (heterodimer formation). This hypothesis was recently investigated in COS-7 cells where fluorescent recombinant eEF1A isoforms colocalized at the level of cytoplasm with a FRET signal more intense at plasma membrane level. Here, we addressed our attention in highlighting and confirming this interaction in a different cell line, HEK 293, normally expressing eEF1A1 but lacking the eEF1A2 isoform. To this end, His-tagged eEF1A2 was expressed in HEK 293 cells and found to colocalize with endogenous eEF1A1 in the cyto - plasm, also at the level of cellular membranes. Moreover, FRET analysis showed, in this case, the appearance of a stronger signal mainly at the level of the plasma membrane. These results confirmed what was previously observed in COS-7 cells and strongly rein forced the interaction among eEF1A isoforms. Moreover, the formation of eEF1A het- erodimer in cancer cells could also be important for cytoskeleton rearrangements rather than for phosphorylation, most likely occurring during cell survival and apoptosis.
{"title":"Cellular Interaction of Human Eukaryotic Elongation Factor 1A Isoforms","authors":"N. Migliaccio, Gennaro Sanità, I. Ruggiero, N. Martucci, C. Sanges, E. Rippa, V. Quagliariello, F. Papale, P. Arcari, A. Lamberti","doi":"10.5772/INTECHOPEN.74733","DOIUrl":"https://doi.org/10.5772/INTECHOPEN.74733","url":null,"abstract":"Besides its canonical role in protein synthesis, the eukaryotic translation elongation fac- tor 1A (eEF1A) is also involved in many other cellular processes such as cell survival and apoptosis. We showed that eEF1A phosphorylation by C-Raf in vitro occurred only in the presence of eEF1A1 and eEF1A2, thus suggesting that both isoforms interacted in cancer cells (heterodimer formation). This hypothesis was recently investigated in COS-7 cells where fluorescent recombinant eEF1A isoforms colocalized at the level of cytoplasm with a FRET signal more intense at plasma membrane level. Here, we addressed our attention in highlighting and confirming this interaction in a different cell line, HEK 293, normally expressing eEF1A1 but lacking the eEF1A2 isoform. To this end, His-tagged eEF1A2 was expressed in HEK 293 cells and found to colocalize with endogenous eEF1A1 in the cyto - plasm, also at the level of cellular membranes. Moreover, FRET analysis showed, in this case, the appearance of a stronger signal mainly at the level of the plasma membrane. These results confirmed what was previously observed in COS-7 cells and strongly rein forced the interaction among eEF1A isoforms. Moreover, the formation of eEF1A het- erodimer in cancer cells could also be important for cytoskeleton rearrangements rather than for phosphorylation, most likely occurring during cell survival and apoptosis.","PeriodicalId":273381,"journal":{"name":"Protein-Protein Interaction Assays","volume":"57 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"121044580","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-07-18DOI: 10.5772/INTECHOPEN.75520
K. Malik, H. Sadia, M. Basit
The generation of genetically modified (GM) crops is rapidly expanding each and every year around the world. The well-being and quality assessment of these harvests are vital issues with respect to buyers’ interests. This drove the administrative specialists to execute an arrangement of extremely strict strategies for the endorsement to develop and use GMOs and to produce an interest in scientific techniques equipped for identifying GM crops. The GM crops have been added to the effective fuse of various attributes by presenting transgenes, for example, Bacillus thuringiensis (Bt) insecticidal qualities, in various crop species. GM crops give critical financial, natural, well-being and social advantages to both small and large agriculturists. The detection strategies incorporate either DNA-based or protein-based measures. Different immunoassays or catalyst connected immunosorbent tests are delicate and more affordable; however, they need experienced technicians. A very simple method, that is, immunochromatographic (ICS) test, is set up in the world, which is modest, compact and simple to utilize. The ICS is a semiquantitative method for indicative screening and semi-measurement of new remote proteins presented through hereditary change of plants. The strip is the easiest method for the assessment of several Bt crop plants for insecticidal quality.
{"title":"Protein-Based Detection Methods for Genetically Modified Crops","authors":"K. Malik, H. Sadia, M. Basit","doi":"10.5772/INTECHOPEN.75520","DOIUrl":"https://doi.org/10.5772/INTECHOPEN.75520","url":null,"abstract":"The generation of genetically modified (GM) crops is rapidly expanding each and every year around the world. The well-being and quality assessment of these harvests are vital issues with respect to buyers’ interests. This drove the administrative specialists to execute an arrangement of extremely strict strategies for the endorsement to develop and use GMOs and to produce an interest in scientific techniques equipped for identifying GM crops. The GM crops have been added to the effective fuse of various attributes by presenting transgenes, for example, Bacillus thuringiensis (Bt) insecticidal qualities, in various crop species. GM crops give critical financial, natural, well-being and social advantages to both small and large agriculturists. The detection strategies incorporate either DNA-based or protein-based measures. Different immunoassays or catalyst connected immunosorbent tests are delicate and more affordable; however, they need experienced technicians. A very simple method, that is, immunochromatographic (ICS) test, is set up in the world, which is modest, compact and simple to utilize. The ICS is a semiquantitative method for indicative screening and semi-measurement of new remote proteins presented through hereditary change of plants. The strip is the easiest method for the assessment of several Bt crop plants for insecticidal quality.","PeriodicalId":273381,"journal":{"name":"Protein-Protein Interaction Assays","volume":"44 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130363883","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-04-07DOI: 10.5772/INTECHOPEN.75644
Y. Ohmuro-Matsuyama, H. Ueda
Protein-fragment complementation assays (PCAs) are commonly used to assay protein–pro- tein interaction (PPI). While PCAs based on firefly luciferase (Fluc) in cells or lysates are a user-friendly method giving a high signal/background (S/B) ratio, they are difficult to use in vitro owing to the instability of split Fluc fragments. As a solution to this issue, we devel oped a novel protein–protein interaction assay named FlimPIA using two mutant Flucs, each of which catalyzes one of the two half-reactions catalyzed by the wild-type enzyme. Upon approximation by the tethered protein pairs, the two mutants yielded higher signal owing to a more efficient transfer of the reaction intermediate luciferyl adenylate. FlimPIA showed many advantages over in vitro split Fluc assays, such as longer detectable distance, more sta - ble probes, and higher signal readout in a shorter time period, and it also worked in cellulo.
{"title":"A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction","authors":"Y. Ohmuro-Matsuyama, H. Ueda","doi":"10.5772/INTECHOPEN.75644","DOIUrl":"https://doi.org/10.5772/INTECHOPEN.75644","url":null,"abstract":"Protein-fragment complementation assays (PCAs) are commonly used to assay protein–pro- tein interaction (PPI). While PCAs based on firefly luciferase (Fluc) in cells or lysates are a user-friendly method giving a high signal/background (S/B) ratio, they are difficult to use in vitro owing to the instability of split Fluc fragments. As a solution to this issue, we devel oped a novel protein–protein interaction assay named FlimPIA using two mutant Flucs, each of which catalyzes one of the two half-reactions catalyzed by the wild-type enzyme. Upon approximation by the tethered protein pairs, the two mutants yielded higher signal owing to a more efficient transfer of the reaction intermediate luciferyl adenylate. FlimPIA showed many advantages over in vitro split Fluc assays, such as longer detectable distance, more sta - ble probes, and higher signal readout in a shorter time period, and it also worked in cellulo.","PeriodicalId":273381,"journal":{"name":"Protein-Protein Interaction Assays","volume":"39 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116087246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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