Статті в журналах: "Recombinant proteins Analysis"

1

Southan, Christopher. "Purification and analysis of recombinant proteins." Trends in Biotechnology 10 (1992): 226. http://dx.doi.org/10.1016/0167-7799(92)90226-l.

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2

Kermasha, S., and I. Alli. "Purification and analysis of recombinant proteins." Food Research International 26, no. 2 (January 1993): 158–59. http://dx.doi.org/10.1016/0963-9969(93)90072-q.

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3

Kaufman, Randal J. "Mammalian recombinant proteins: Structure, function and immunological analysis." Current Opinion in Biotechnology 1, no. 2 (December 1990): 141–50. http://dx.doi.org/10.1016/0958-1669(90)90023-e.

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4

Senear, Donald F., Robert A. Mendelson, Deborah B. Stone, Linda A. Luck, Elena Rusinova, and J. B. Alexander Ross. "Quantitative Analysis of Tryptophan Analogue Incorporation in Recombinant Proteins." Analytical Biochemistry 300, no. 1 (January 2002): 77–86. http://dx.doi.org/10.1006/abio.2001.5441.

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5

Kollárovič, G., D. Majera, K. Luciaková, and P. Baráth. "Expression and purification of recombinant NFI proteins for functional analysis." General Physiology and Biophysics 28, no. 4 (2009): 331–39. http://dx.doi.org/10.4149/gpb_2009_04_331.

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6

Coutinho, M., K. S. Aulak, and A. E. Davis. "Functional analysis of the serpin domain of C1 inhibitor." Journal of Immunology 153, no. 8 (October 15, 1994): 3648–54. http://dx.doi.org/10.4049/jimmunol.153.8.3648.

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Abstract To analyze the role of the heavily glycosylated amino-terminal domain of C1 inhibitor in protease inhibitory activity, two truncated C1 inhibitor molecules were constructed. The abilities of the recombinant truncated inhibitors to complex with target proteases were compared with that of the wild-type recombinant protein. One recombinant truncated molecule consisted of amino acid residues 76 to 478 (C-serp(76)) and the other of residues 98 to 478 (C-serp(98)). The recombinant proteins were each expressed in similar quantities. The thermal denaturation profiles of the two truncated proteins were similar to that of the wild-type protein. Identical binding of C1s, C1r, kallikrein, and beta factor XIIa was observed with the three molecules. Furthermore, the truncated molecules also effectively inhibited C1 activity in hemolytic assays. These studies therefore clearly demonstrate that the amino-terminal domain of C1 inhibitor does not influence complex formation with target proteases.

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7

Jerlström-Hultqvist, Jon, Britta Stadelmann, Sandra Birkestedt, Ulf Hellman, and Staffan G. Svärd. "Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome." Eukaryotic Cell 11, no. 7 (May 18, 2012): 864–73. http://dx.doi.org/10.1128/ec.00092-12.

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ABSTRACTIn recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoanGiardia intestinaliscan be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use inGiardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathioneS-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinantGiardiaproteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of theG. intestinalis26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and fromGiardia, which will allow the study of specific parasite proteins and protein complexes.

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8

Sims, Andrew H., Manda E. Gent, Karin Lanthaler, Nigel S. Dunn-Coleman, Stephen G. Oliver, and Geoffrey D. Robson. "Transcriptome Analysis of Recombinant Protein Secretion by Aspergillus nidulans and the Unfolded-Protein Response In Vivo." Applied and Environmental Microbiology 71, no. 5 (May 2005): 2737–47. http://dx.doi.org/10.1128/aem.71.5.2737-2747.2005.

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ABSTRACT Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.

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9

Zeck, Anne, Jörg Thomas Regula, Vincent Larraillet, Björn Mautz, Oliver Popp, Ulrich Göpfert, Frank Wiegeshoff, et al. "Low Level Sequence Variant Analysis of Recombinant Proteins: An Optimized Approach." PLoS ONE 7, no. 7 (July 6, 2012): e40328. http://dx.doi.org/10.1371/journal.pone.0040328.

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10

Viseux, N., X. Hronowski, J. Delaney, and B. Domon. "Qualitative and Quantitative Analysis of the Glycosylation Pattern of Recombinant Proteins." Analytical Chemistry 73, no. 20 (October 2001): 4755–62. http://dx.doi.org/10.1021/ac015560a.

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11

Coulot, M., B. Domon, H. Grossenbacher, C. Guenat, W. Maerki, D. R. Müller, and W. J. Richter. "LC-MS and MS/MS in the analysis of recombinant proteins." Journal of Molecular Structure 292 (March 1993): 89–103. http://dx.doi.org/10.1016/0022-2860(93)80092-a.

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12

Steen, Johanna, Margareta Ramström, Mathias Uhlén, Sophia Hober, and Jenny Ottosson. "Automated sample preparation method for mass spectrometry analysis on recombinant proteins." Journal of Chromatography A 1216, no. 20 (May 2009): 4457–64. http://dx.doi.org/10.1016/j.chroma.2009.03.041.

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13

Bischoff, Rainer, Dominique Roecklin, and Carolyn Roitsch. "Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients." Electrophoresis 13, no. 1 (1992): 214–19. http://dx.doi.org/10.1002/elps.1150130144.

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14

Fei, Dongliang, Yaxi Guo, Qiong Fan, Ming Li, Li Sun, Mingxiao Ma, and Yijing Li. "Codon optimization, expression in Escherichia coli, and immunogenicity analysis of deformed wing virus (DWV) structural protein." PeerJ 8 (March 11, 2020): e8750. http://dx.doi.org/10.7717/peerj.8750.

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Background Deformed wing virus (DWV) is a serious threat to honey bees (Apis mellifera) and is considered a major cause of elevated losses of honey bee colonies. However, lack of information on the immunogenicity of DWV structural proteins has hindered the development of effective biocontrol drugs. Methods We optimized the VP1, VP2 and VP3 codons of DWV surface capsid protein genes on the basis of an Escherichia coli codon bias, and the optimized genes of roVP1, roVP2 and roVP3 were separately expressed in E. coli and purified. Next, the three recombinant proteins of roVP1, roVP2 and roVP3 were intramuscularly injected into BALB/c and the immunogenicity was evaluated by the levels of specific IgG and cytokines. Furthermore, anti-roVP-antisera (roVP1 or roVP2 or roVP3) from the immunized mice was incubated with DWV for injecting healthy white-eyed pupae for the viral challenge test, respectively. Results The optimized genes roVP1, roVP2 and roVP3 achieved the expression in E. coli using SDS-PAGE and Western blotting. Post-immunization, roVP2 and roVP3 exhibited higher immunogenicity than roVP1 and stimulated a stronger humoral immune response in the mice, which showed that the recombinant proteins of roVP3 and roVP2 induced a specific immune response in the mice. In the challenge test, data regarding quantitative real-time RT-PCR (qRT-PCR) from challenged pupae showed that the level of virus copies in the recombinant protein groups was significantly lower than that of the virus-only group at 96 h post-inoculation (P < 0.05). Among them, the degree of neutralization using antibodies raised to the recombinant proteins are between approximately 2-fold and 4-fold and the virus copies of the roVP3 group are the lowest in the three recombinant protein groups, which indicated that specific antibodies against recombinant proteins roVP1, roVP2 and roVP3 of DWV could neutralize DWV to reduce the virus titer in the pupae. Collectively, these results demonstrated that the surface capsid protein of DWV acted as candidates for the development of therapeutic antibodies against the virus.

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15

Pachner, A. R., D. Dail, L. Li, L. Gurey, S. Feng, E. Hodzic, and S. Barthold. "Humoral Immune Response Associated with Lyme Borreliosis in Nonhuman Primates: Analysis by Immunoblotting and Enzyme-Linked Immunosorbent Assay with Sonicates or Recombinant Proteins." Clinical and Vaccine Immunology 9, no. 6 (November 2002): 1348–55. http://dx.doi.org/10.1128/cdli.9.6.1348-1355.2002.

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ABSTRACT The immune response to Borrelia burgdorferi, the causative agent of Lyme disease, is complex. We studied the immunoglobulin M (IgM) and IgG antibody response to N40Br, a sensu stricto strain, in the rhesus macaque(nonhuman primate [NHP]) model of infection to identify the spirochetal protein targets of specific antibody. Antigens used in enzyme-linked immunosorbent assays were whole-cell sonicates of the spirochete and recombinant proteins of B. burgdorferi. Immunoblotting with a commercially available strip and subsequent quantitative densitometry of the bands were also used. Sera from four different groups of NHPs were used: immunocompetent, transiently immunosuppressed, extended immunosuppressed, and uninfected. In immunocompetent and transiently immunosuppressed NHPs, there was a strong IgM and IgG response. Major proteins for the early IgM response were P39 and P41 and recombinant BmpA and OspC. Major proteins for the later IgG response were P39, P41, P18, P60, P66, and recombinant BmpA and DbpA. There was no significant response in the NHPs to recombinant OspA or to Arp, a 37-kDa protein that elicits an antibody response during infection in mice. Most antibody responses, except for that to DbpA, were markedly diminished by prolonged dexamethasone treatment. This study supports the hypothesis that recombinant proteins may provide a useful adjunct to current diagnostic testing for Lyme borreliosis.

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16

Shi, Xiaohong, and Richard M. Elliott. "Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins." Journal of General Virology 90, no. 2 (February 1, 2009): 297–306. http://dx.doi.org/10.1099/vir.0.007567-0.

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The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.

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17

Karavaev, VS, E. S. Oleinikova, M. Sh Azaev, and A. B. Beklemishev'. "IMMUNOCHEMICAL ANALYSIS OF RECOMBINANT CHIMERIC POLYPEPTIDE OspCg_ar+afz OF BORRELIA GARINII AND B. AFZELIIISOLATES." Journal of microbiology, epidemiology and immunobiology, no. 3 (June 28, 2016): 37–44. http://dx.doi.org/10.36233/0372-9311-2016-3-37-44.

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Aim. Comparative study of antigenic properties of recombinant proteins OspCgar and OspCafz and recombinant chimeric polypeptide OspCgar+afZ, that contains amino acid sequences of mature immune dominant OspC proteins of West-Siberian isolates of Borrelia garinii (OspCgar) and B. afzelii (OspCafz), and evaluation of possibility of their use as antigens during creation of test-systems for serodiagnostics of Lyme borreliosis (LB) on the territory of Western Siberia. Materials and methods. Recombinant chimeric polypeptide OspCgar+afz and recombinant mature proteins OspCgar and OspCafz, obtained by expression of the corresponding genes in Escherichia coli cells, purified by affinity chromatography in Ni-NTA-sepharose CL-6B and studied by EIA method for the ability to bind antibodies from sera of LB patients. Results. A difference in sensitivity of determination by EIA method of specific IgM and IgG against borreliae in blood sera of LB patients with localized stage of the disease during use of OspCgar, OspCafz and OspCgar+afZ chimera as antigens was shown. Chimeric antigen OspCgar+afz was established to show higher antigenic activity compared with each of the OspCgar or OspCafZ antigens separately. Conclusion. The results of the study allow to examine the recombinant chimeric polypeptide OspCgar+afz as a possible component during creation of test-systems for serodiagnostics of LB on the territory of West Siberia.

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18

Oulton, Tate, Joshua Obiero, Isabel Rodriguez, Isaac Ssewanyana, Rebecca A. Dabbs, Christine M. Bachman, Bryan Greenhouse, et al. "Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray." PLOS ONE 17, no. 8 (August 29, 2022): e0273106. http://dx.doi.org/10.1371/journal.pone.0273106.

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The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro transcription/translation (IVTT) systems–a similarly high-throughput protein expression method–are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on the same protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from different expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clear correlation between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.

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19

Tu, Chien, Ruey-Yu Chiou, and Meei-Mei Chen. "CLONING, EXPRESSION AND PRELIMINARY ANTIGENICITY ANALYSIS OF STRUCTURAL PROTEINS OF A KOI HERPESVIRUS ISOLATE FROM KOI, CYPRINUS CARPIO IN TAIWAN." Taiwan Veterinary Journal 40, no. 02 (June 2014): 69–75. http://dx.doi.org/10.1142/s1682648514500097.

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The aim of this study was to clone and express the ORF72 and ORF92 genes of koi herpesvirus (KHV) in a prokaryotic system and to examine the antigenicity of recombinant proteins. Phylogenetic analysis revealed that both ORF72 and ORF92 had 100% homology with KHV-J, and 99% homology with those from KHV-U and KHV-I in nucleotides. This suggests that the KHV isolate in Taiwan is more closely related to the Japanese strain (Asian genotype). In the antigenicity analysis, the crude recombinant ORF72 and ORF92 capsid proteins reacted with the positive sera of the survival fish after a KHV outbreak, indicating that these recombinant capsid proteins might mimic antigens of the wild type KHV to induce an immunological response in the infected host. Our results demonstrated potential for general applicability to serological tests and vaccine development.

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20

Grover, J., and P. J. Roughley. "The expression of functional link protein in a baculovirus system: analysis of mutants lacking the A, B and B' domains." Biochemical Journal 300, no. 2 (June 1, 1994): 317–24. http://dx.doi.org/10.1042/bj3000317.

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Functional recombinant human link protein has been produced using a baculovirus expression system. In addition to the intact link protein, three mutant forms have also been expressed. Each mutant bears a deletion equivalent to the protein encoded by one exon in the gene. These deletions represent the A domain, which is thought to be responsible for interaction with aggrecan, and the B or B' domains, which are associated with the interaction with hyaluronate. Such deletions split codons spanning exon boundaries, but maintain the reading frame of the protein and result in the correct amino acid being present at the splice junction. All the recombinant proteins appear as two components upon SDS/PAGe, though the abundance of the two forms does vary between preparations, as a result of variable substitution by N-linked oligosaccharides. The recombinant intact link protein was able to interact with both hyaluronate and aggrecan, showing that the baculovirus system is able to produce functional molecules. All of the recombinant mutant link proteins were also able to interact with hyaluronate, indicating that both the B and B' domains can function independently. The recombinant mutant link proteins were also able to interact with aggrecan, with the exception of the mutant lacking the A domain, confirming that this ability resides entirely within this domain.

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21

Bhandari, Bikash K., Chun Shen Lim, Daniela M. Remus, Augustine Chen, Craig van Dolleweerd, and Paul P. Gardner. "Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites." PLOS Computational Biology 17, no. 10 (October 5, 2021): e1009461. http://dx.doi.org/10.1371/journal.pcbi.1009461.

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Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann’s ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during overexpression.

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22

Santos, R. B., A. S. Pires, H. S. Silva, and and R. Abranches. "Assessment of Medicago Based Systems for the Production of Human Proteins: Microscopy Analysis of the Subcellular Deposition Patterns of the Recombinant Product." Microscopy and Microanalysis 18, S5 (August 2012): 11–12. http://dx.doi.org/10.1017/s1431927612012718.

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The use of transgenic plants for the large scale production of recombinant proteins with commercial and therapeutic value has emerged as an alternative to conventional platforms. Plant based systems, including whole plants and plant cell cultures offer many advantages particularly regarding safety and cost effectiveness. In our laboratory we have been using the model plant Medicago truncatula as a system to express recombinant proteins with a variety of applications.

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23

Guo, Rong, Shiguang Liu, Robert F. Spurney, and L. D. Quarles. "Analysis of recombinant Phex: an endopeptidase in search of a substrate." American Journal of Physiology-Endocrinology and Metabolism 281, no. 4 (October 1, 2001): E837—E847. http://dx.doi.org/10.1152/ajpendo.2001.281.4.e837.

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X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (r Phex-WT) and inactive mutant Phex proteins (r Phex-3′M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172–186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by r Phex-WT. In addition, membranes expressing r Phex-WT, r Phex-3′M, and the empty vector hydrolyzed parathyroid hormone-(1–34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, r Phex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant r Phex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

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Horvath, C. M., A. Wolven, D. Machadeo, J. Huber, L. Boter, M. Benedetti, B. Hempstead, and M. V. Chao. "Analysis of the trk NGF receptor tyrosine kinase using recombinant fusion proteins." Journal of Cell Science 1993, Supplement 17 (December 1, 1993): 223–28. http://dx.doi.org/10.1242/jcs.1993.supplement_17.31.

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25

TOTTÉ, PHILIPPE, DECLAN McKEEVER, FRANS JONGEJAN, ANTONY BARBET, SUMAN M. MAHAN, DUNCAN MWANGI, and ALBERT BENSAID. "Analysis of Cellular Responses to Native and Recombinant Proteins of Cowdria ruminantiumaa." Annals of the New York Academy of Sciences 849, no. 1 (June 1998): 155–60. http://dx.doi.org/10.1111/j.1749-6632.1998.tb11045.x.

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26

Lim, Kwang Suk, Young-Wook Won, Yong Soo Park, and Yong-Hee Kim. "Preparation and functional analysis of recombinant protein transduction domain-metallothionein fusion proteins." Biochimie 92, no. 8 (August 2010): 964–70. http://dx.doi.org/10.1016/j.biochi.2010.04.005.

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27

Rehbein, Peter, Krishna Saxena, Kai Schlepckow, and Harald Schwalbe. "Protocol for aerosol-free recombinant production and NMR analysis of prion proteins." Journal of Biomolecular NMR 59, no. 2 (April 26, 2014): 111–17. http://dx.doi.org/10.1007/s10858-014-9831-5.

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28

Kotelnikova, O. V., A. A. Zinchenko, A. A. Vikhrov, A. P. Alliluev, O. V. Serova, E. A. Gordeeva, L. S. Zhigis, et al. "Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins." Bulletin of Experimental Biology and Medicine 161, no. 3 (July 2016): 391–94. http://dx.doi.org/10.1007/s10517-016-3422-2.

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29

Murrell, J. R., R. G. Schoner, J. J. Liepnieks, H. N. Rosen, A. C. Moses, and M. D. Benson. "Production and functional analysis of normal and variant recombinant human transthyretin proteins." Journal of Biological Chemistry 267, no. 23 (August 1992): 16595–600. http://dx.doi.org/10.1016/s0021-9258(18)42044-3.

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30

Nguyen, Khanh Tan, Hung Manh Tran, Anh Tuan Trieu, Van Thi Huynh Nguyen, Hoang M. Nguyen, and Phu Tran Vinh Phu. "PURIFICATION AND ANALYSIS CATALYTIC FUNCTIONS OF RECOMBINANT INFLUENZA VIRAL POLYMERASES." Journal of microbiology, biotechnology and food sciences 11, no. 4 (February 1, 2022): e4954. http://dx.doi.org/10.55251/jmbfs.4954.

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Polymerase of influenza virus is made up of three subunits PB1, PB2, and PA, which are involved in viral genome transcription and replication. Purification of sufficient amounts of viral polymerase is essential to understand the catalytic function of viral polymerase. In this study, we generated a viral polymerase expression system in human embryonic kidney 293T cells (293T cells). The cDNAs for RNA segments 1, 2, and 3, which encode for PB2, PB1, and PA proteins, respectively, were integrated into the mammalian expression plasmids pCAGGS to simultaneously express all viral polymerase proteins in 293T cells. We purified the recombinant polymerases of human influenza virus A/PR/8/34 (H1N1) (PR8) and avian influenza virus A/Turkey/England/1969 (H3N2) (TE) using anti-FLAG M2 affinity resin. After confirming trimeric complexes, enzymatic properties of recombinant polymerases were characterized, including model viral RNA binding, in vitro transcription assays, and cap-snatching activity upon addition of cap1-70 mer vRNA as substrate. Taken together we conclude that 293T cells are a suitable expression system for sufficient amount isolation of functional recombinant influenza viral polymerases with 95% of purity.

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Jaleel, Zaroug, Shun Zhou, Zaira Martín-Moldes, Lauren M. Baugh, Jonathan Yeh, Nina Dinjaski, Laura T. Brown, Jessica E. Garb, and David L. Kaplan. "Expanding Canonical Spider Silk Properties through a DNA Combinatorial Approach." Materials 13, no. 16 (August 14, 2020): 3596. http://dx.doi.org/10.3390/ma13163596.

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The properties of native spider silk vary within and across species due to the presence of different genes containing conserved repetitive core domains encoding a variety of silk proteins. Previous studies seeking to understand the function and material properties of these domains focused primarily on the analysis of dragline silk proteins, MaSp1 and MaSp2. Our work seeks to broaden the mechanical properties of silk-based biomaterials by establishing two libraries containing genes from the repetitive core region of the native Latrodectus hesperus silk genome (Library A: genes masp1, masp2, tusp1, acsp1; Library B: genes acsp1, pysp1, misp1, flag). The expressed and purified proteins were analyzed through Fourier Transform Infrared Spectrometry (FTIR). Some of these new proteins revealed a higher portion of β-sheet content in recombinant proteins produced from gene constructs containing a combination of masp1/masp2 and acsp1/tusp1 genes than recombinant proteins which consisted solely of dragline silk genes (Library A). A higher portion of β-turn and random coil content was identified in recombinant proteins from pysp1 and flag genes (Library B). Mechanical characterization of selected proteins purified from Library A and Library B formed into films was assessed by Atomic Force Microscopy (AFM) and suggested Library A recombinant proteins had higher elastic moduli when compared to Library B recombinant proteins. Both libraries had higher elastic moduli when compared to native spider silk proteins. The preliminary approach demonstrated here suggests that repetitive core regions of the aforementioned genes can be used as building blocks for new silk-based biomaterials with varying mechanical properties.

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Bakli, Mahfoud, Raul Pascalau, and Laura Smuleac. "Rare Codon Analysis in Rickettsia Affecting Recombinant Protein Expression in Escherichia coli." Advanced Research in Life Sciences 4, no. 1 (January 1, 2020): 30–35. http://dx.doi.org/10.2478/arls-2020-0015.

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Abstract Rickettsia species are important emerging pathogens causing rickettsial diseases, which are important cause death worldwide. The number of recombinant proteins used for diagnostic and therapeutic applications has increased dramatically, which is important in determination of protein function, structure and antigensity. Although E. coli is widely used expression system, the codon bias can hamper protein expression due to the presence of rare codons in gene sequence coding protein of interest. Using bioinformatics tools, rare codon analysis of rickettsial genes was performed and compared to not expressed proteins in both R. prowazekii and R. rickettsii. A negative correlation between frequencies of rare codons in Rickettsia and success of rickettsial protein expression was observed. This study suggested a useful tool to improve rickettsial recombinant protein expression in E. coli.

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McBride, Jere W., Lucy M. Ndip, Vsevolod L. Popov, and David H. Walker. "Identification and Functional Analysis of an Immunoreactive DsbA-Like Thio-Disulfide Oxidoreductase of Ehrlichia spp." Infection and Immunity 70, no. 5 (May 2002): 2700–2703. http://dx.doi.org/10.1128/iai.70.5.2700-2703.2002.

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ABSTRACT Novel homologous DsbA-like disulfide bond formation (Dsb) proteins of Ehrlichia chaffeensis and Ehrlichia canis were identified which restored DsbA activity in complemented Escherichia coli dsbA mutants. Recombinant Ehrlichia Dsb (eDsb) proteins were recognized by sera from E. canis-infected dogs but not from E. chaffeensis-infected patients. The eDsb proteins were observed primarily in the periplasm of E. chaffeensis and E. canis.

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Koupriyanov, V. V., L. I. Nikolaeva, A. A. Zykova, P. I. Makhnovskiy, R. Y. Kotlyarov, A. V. Vasilyev, and N. V. Ravin. "IMMUNOGENIC PROPERTIES OF RECOMBINANT MOZAIC PROTEINS BASED ON ANTIGENS NS4A AND NS4B OF HEPATITIS C VIRUS." Problems of Virology, Russian journal 63, no. 3 (June 20, 2018): 138–43. http://dx.doi.org/10.18821/0507-4088-2018-63-3-138-143.

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The aim of the study was to investigate immunogenic properties of mosaic recombinant proteins constructed on the data of hepatitis C virus NS4A and NS4B antigens. Four mosaic recombinant proteins, containing the T and B epitopes of the NS4A and NS4B antigens, were created by genetic engineering methods in the E. coli system. To enhance the immune response they were linked in different variations to the nucleotide sequences of murine interleukin-2 (IL-2), the Neisseria meningiditis lipopeptide, and the T helper epitope of the core protein of hepatitis C virus. The immunogenic properties of these recombinant proteins were analyzed by immunoblotting, ELISA and ELISpot using sera from immunized mice and patients infected with hepatitis C virus. Recombinant proteins specifically reacted with the sera of immunized mice and infected patients in immunoblotting. According to the ELISA data, the predominant formation of antibodies to NS4B was observed when mice were immunized with the recombinant proteins containing both antigens. Analysis of gamma-interferon production by T-lymphocytes upon contact with activated dendritic cells showed in ELISpot that the maximum production of this cytokine was detected when adjuvant components were located at the N- and C-ends of the recombinant protein. The highest level of gamma-interferon production during stimulation with this drug was detected in lymphocytes from the bone marrow and lymph nodes. The recombinant protein containing the T and B epitopes of NS4A and NS4B, murine IL-2 and the lipopeptide Neisseria meningiditis had the greatest immunostimulate effect among the four constructions. This recombinant protein formed nanoparticles of 100-120 nm in size.

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Okenu, Daniel M. N., Eleanor M. Riley, Quentin D. Bickle, Philip U. Agomo, Arnoldo Barbosa, Jon R. Daugherty, David E. Lanar, and David J. Conway. "Analysis of Human Antibodies to Erythrocyte Binding Antigen 175 of Plasmodium falciparum." Infection and Immunity 68, no. 10 (October 1, 2000): 5559–66. http://dx.doi.org/10.1128/iai.68.10.5559-5566.2000.

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ABSTRACT Invasion of human erythrocytes by Plasmodium falciparummerozoites is a multistep process. For many strains of the parasite, part of this process requires that the erythrocyte binding antigen 175 (EBA-175) of the merozoite binds to sialic acid residues of glycophorin A on the erythrocyte surface, a receptor-ligand interaction which represents a potential target for inhibition by antibodies. This study characterizes the reactivity of naturally acquired human antibodies with four recombinant proteins representing parts of EBA-175 (region II, regions III to V, and the dimorphic C and F segment region) in populations in which the organism is endemic. Serum immunoglobulin G (IgG) recognizing the recombinant proteins is predominantly of the IgG1 and IgG3 subclasses, and its prevalence increases with age. In a large population study in The Gambia, serum positivity for IgG or IgG1 and IgG3 subclass antibodies to each of the EBA-175 recombinant antigens was not significantly associated with subsequent protection from clinical malaria. However, there was a trend indicating that individuals with high levels of IgG to region II may have some protection.

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Hamsten, Carl, Georgina Tjipura-Zaire, Laura McAuliffe, Otto J. B. Huebschle, Massimo Scacchia, Roger D. Ayling, and Anja Persson. "Protein-Specific Analysis of Humoral Immune Responses in a Clinical Trial for Vaccines against Contagious Bovine Pleuropneumonia." Clinical and Vaccine Immunology 17, no. 5 (March 31, 2010): 853–61. http://dx.doi.org/10.1128/cvi.00019-10.

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ABSTRACT Specific humoral immune responses in a clinical trial on cattle for vaccines against contagious bovine pleuropneumonia (CBPP) were investigated. The trial included a subunit vaccine consisting of five recombinant putative variable surface proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) compared to the currently approved attenuated vaccine strain T1/44 and untreated controls. Humoral immune responses to 65 individual recombinant surface proteins of M. mycoides SC were monitored by a recently developed bead-based array assay. Responses to the subunit vaccine components were found to be weak. Animals vaccinated with this vaccine were not protected and had CBPP lesions similar to those of the untreated controls. In correlating protein-specific humoral responses to T1/44-induced immunity, five proteins associated with a protective immune response were identified by statistical evaluation, namely, MSC_1046 (LppQ), MSC_0271, MSC_0136, MSC_0079, and MSC_0431. These five proteins may be important candidates in the development of a novel subunit vaccine against CBPP.

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Deykin, Alexey V., Olesya V. Shcheblykina, Elena E. Povetka, Polina A. Golubinskaya, Vladimir M. Pokrovsky, Liliya V. Korokina, Olesya A. Vanchenko, et al. "Genetically modified animals for use in biopharmacology: from research to production." Research Results in Pharmacology 7, no. 4 (October 29, 2021): 11–27. http://dx.doi.org/10.3897/rrpharmacology.7.76685.

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Introduction: In this review, the analysis of technologies for obtaining biologically active proteins from various sources is carried out, and the comparative analysis of technologies for creating producers of biologically active proteins is presented. Special attention is paid to genetically modified animals as bioreactors for the pharmaceutical industry of a new type. The necessity of improving the technology of development transgenic rabbit producers and creating a platform solution for the production of biological products is substantiated. The advantages of using TrB for the production of recombinant proteins: The main advantages of using TrB are the low cost of obtaining valuable complex therapeutic human proteins in readily accessible fluids, their greater safety relative to proteins isolated directly from human blood, and the greater safety of the activity of the native protein. The advantages of the mammary gland as a system for the expression of recombinant proteins: The mammary gland is the organ of choice for the expression of valuable recombinant proteins because milk is easy to collect in large volumes. Methods for obtaining transgenic animals: The modern understanding of the regulation of gene expression and the discovery of new tools for gene editing can increase the efficiency of creating bioreactors for animals and help to obtain high concentrations of the target protein. The advantages of using rabbits as bioreactors producing recombinant proteins in milk: The rabbit is a relatively small animal with a short duration of gestation, puberty and optimal size, capable of producing up to 5 liters of milk per year per female, receiving up to 300 grams of the target protein.

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38

Mahadevappa, Mamatha, Richard A. DeScenzo, and Roger P. Wise. "Recombination of alleles conferring specific resistance to powdery mildew at the Mla locus in barley." Genome 37, no. 3 (June 1, 1994): 460–68. http://dx.doi.org/10.1139/g94-064.

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In barley (Hordeum vulgare L.), the Mla locus conditions reaction to the powdery mildew fungus Erysiphe graminis f.sp. hordei. Enrichment for genetic recombinants in the Mla region is possible by screening for recombination events between the flanking endosperm storage proteins hordeins C and B. Reciprocal crosses were made between the Franger (C.I. 16151) and Rupee (C.I. 16155) lines carrying the (Mla6 + Mla14) and Mla13 alleles, respectively. Recombinants were identified from F2 segregants by analyzing the extracted hordein polypeptides by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. Two hundred and seventy-six recombinant gametes were identified from the 1800 seeds that were screened. Recombination of Mla alleles was analyzed by inoculating F4 recombinant lines with three isolates of E. graminis (A27, 5874, and CR3), which recognize specific Mla alleles. The linkage order established is Hor1–Mla6–Mla13–Mla14–Hor2. The genetic distances between Hor1–Mla6, Mla6–Mla13, and Mla13–Hor2, obtained using Mapmaker 3.0b F3 intercross analysis, are 3.9, 0.2, and 5.2 cM, respectively.Key words: recombinant, barley, powdery mildew, Mla, hordein.

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39

Fernandes, Luis G. V., Monica L. Vieira, Ivy J. Alves, Zenaide M. de Morais, Silvio A. Vasconcellos, Eliete C. Romero, and Ana L. T. O. Nascimento. "Functional and immunological evaluation of two novel proteins of Leptospira spp." Microbiology 160, no. 1 (January 1, 2014): 149–64. http://dx.doi.org/10.1099/mic.0.072074-0.

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This work shows the production and characterization of two novel putative lipoproteins encoded by the genes LIC10645 and LIC10731 identified in the genome sequences of Leptospira interrogans. In silico conservation analysis indicated that the proteins are well conserved among pathogenic leptospiral serovars and species. Recombinant proteins were obtained in Escherichia coli BL21(DE3) Star pLysS strain, purified by metal-affinity chromatography, and used for characterization and immunological evaluations. Recombinant proteins were capable of eliciting a combination of humoral and cellular immune responses in animal models, and could be recognized by antibodies present in human serum samples. The recombinant proteins Lsa44 and Lsa45 were able to bind laminin, and were named Lsa44 and Lsa45 for leptospiral surface adhesins of 44 and 45 kDa, respectively. The attachment to laminin was dose-responsive with K D values of 108.21 and 250.38 nM for Lsa44 and Lsa45, respectively. Moreover, these proteins interact with plasminogen (PLG) with K D values of 53.56 and 36.80 nM, respectively. PLG bound to the recombinant proteins could be converted to plasmin (PLA) in the presence of an activator. Cellular localization assays suggested that the Lsa44 and Lsa45 were surface-exposed. These are versatile proteins capable of interacting with laminin and PLG/PLA, and hence could mediate bacterial adhesion and contribute to tissue penetration.

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40

Wei, Wenrui, Nengxing Shen, Jie Xiao, Yuanyuan Tao, Yuejun Luo, Christiana Angel, Xiaobin Gu, et al. "Expression Analysis and Serodiagnostic Potential of Microneme Proteins 1 and 3 in Eimeria stiedai." Genes 11, no. 7 (June 29, 2020): 725. http://dx.doi.org/10.3390/genes11070725.

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Eimeria stiedai is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant economic losses in the domestic rabbit industry. Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease. Therefore, in the present study we cloned and expressed the two microneme proteins i.e., microneme protein 1 (EsMIC1) and microneme protein 3 (EsMIC3) from E. stiedai and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding EsMIC1 and EsMIC3 were cloned and the mRNA expression levels of these two genes at different developmental stages of E. stiedai were determined by quantitative real-time PCR analysis (qRT-PCR). The immunoreactivity of recombinant EsMIC1 (rEsMIC1) and EsMIC3 (rEsMIC3) proteins were detected by Western blotting, and indirect enzyme-linked immunosorbent assays (ELISAs) based on these two recombinant antigens were established to evaluate their serodiagnostic potential. Our results showed that the proteins encoded by the ORFs of EsMIC1 (711 bp) and EsMIC3 (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both EsMIC1 and EsMIC3 showed the highest mRNA expression levels in the merozoites stage of E. stiedai. Western blotting analysis revealed that both recombinant proteins were recognized by E. stiedai positive sera, and the indirect ELISAs using rEsMIC1 and rEsMIC3 were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity for rEsMIC1 with 100% (48/48) sensitivity and 100% (48/48) specificity for rEsMIC3, respectively. Moreover, rEsMIC1- and rEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 post E. stiedai infection, with the highest positive diagnostic rate (62.5% (30/48) for rEsMIC1 and 66.7% (32/48) for rEsMIC3) observed at day 10 post infection. Therefore, both EsMIC1 and EsMIC3 can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by E. stiedai.

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41

Bidmeshki Barzoki, Tahereh, Ali Mohammad Ahadi, and Hoda Ayat. "A New Design and Epitopes Analysis for Recombinant Vaccine against Salmonella typhi by In silico Analysis." Trends in Immunotherapy 4, no. 2 (August 24, 2020): 1. http://dx.doi.org/10.24294/ti.v4.i2.891.

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Nowadays, foodborne diseases are one of the main problems of the world that infect humans due to consumption of contaminated water or food. Typhoid fever is one of the major causes of illness and death in the world caused by Salmonella typhi. Vaccination is one of the most effective approaches in order to reduction of the disease risk. The main goal of this study is designing and characterization of antigenic determinants of a fusion protein originated from S.typhi usable as an effective vaccine. In this study, the outer membrane proteins of salmonella have been considered as candidates conferring protection against typhoid. Considering the evidence, OmpA, OmpF and OmpC proteins of salmonella applied in a multivalent vaccine design. Conserved motives of these proteins were selected using the CLC software and then their extracellular regions of these peptides were identified with PRED-TMBB server. Appropriate motives were combined for design of final fusion protein. Finally epitops of designed protein with high antigenic properties were identified using BCPREDS, Ellipro, ABCpred, EpiJen, NetCTL-1.2, CTLpred, TAPpred, ProPred and VaxiJen servers. Predicted designed protein in this study reached a very high scores for antigenic indexes. Encoding Genetic construction of this fusion protein could be applied for production of the recombinant OmpA.OmpF.OmpC derived fusion protein with effective antigenic properties as a new vaccine against S.typhi. Laboratory experiments and animal challenging analyses is ongoing.

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Terkawi, Mohamad Alaa, Nguyen Xuan Huyen, Putut Eko Wibowo, Faasoa Junior Seuseu, Mahmoud Aboulaila, Akio Ueno, Youn-Kyoung Goo, Naoaki Yokoyama, Xuenan Xuan, and Ikuo Igarashi. "Spherical Body Protein 4 Is a New Serological Antigen for Global Detection ofBabesia bovisInfection in Cattle." Clinical and Vaccine Immunology 18, no. 2 (December 1, 2010): 337–42. http://dx.doi.org/10.1128/cvi.00388-10.

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ABSTRACTFiveBabesia bovisrecombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentallyB. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentallyB. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions ofB. bovisendemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies toB. bovisin cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.

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Fenton, Brian, and David Walliker. "Genetic analysis of polymorphic proteins of the human malaria parasite Plasmodium falciparum." Genetical Research 55, no. 2 (April 1990): 81–86. http://dx.doi.org/10.1017/s0016672300025301.

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SummaryFive polymorphic proteins, detected by two-dimensional electrophoresis, were analysed in the parents and progeny of a cross between two clones of the malaria parasite Plasmodium falciparum. The information obtained showed that different forms of each protein were determined by allelic variants of each respective gene. One protein was identified as the parasite enzyme adenosine deaminase. Recombinant parasites were produced at a higher than expected frequency.

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44

Rakleova, Gorica, Ivanka Tsacheva, Mima Petkova, Ivelyn Pantchev, and Magdalena Tchorbadjieva. "Generation of Recombinant Antibodies against Orchardgrass Acidic nsLTP-Like Proteins." Zeitschrift für Naturforschung C 63, no. 5-6 (June 1, 2008): 395–402. http://dx.doi.org/10.1515/znc-2008-5-614.

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Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the nonembryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naı¨ve phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.

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45

Annweiler, A., S. Zwilling, R. A. Hipskind, and T. Wirth. "Analysis of transcriptional stimulation by recombinant Oct proteins in a cell-free system." Journal of Biological Chemistry 268, no. 4 (February 1993): 2525–34. http://dx.doi.org/10.1016/s0021-9258(18)53807-2.

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46

Chan, Kun-Wei, Hsien-Hua Hsieh, Hsien-Chi Wang, Ya-Jane Lee, Ming-Hua Sung, Min-Liang Wong, and Wei-Li Hsu. "Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus." Journal of Virological Methods 155, no. 1 (January 2009): 18–24. http://dx.doi.org/10.1016/j.jviromet.2008.09.024.

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47

Eaton, Leslie C. "Quantitation of residual Escherichia coli DNA in recombinant biopharmaceutical proteins by hybridization analysis." Journal of Pharmaceutical and Biomedical Analysis 7, no. 5 (January 1989): 633–38. http://dx.doi.org/10.1016/0731-7085(89)80230-4.

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48

Sako, Yasuhito, Minoru Nakao, Takashi Ikejima, Xian Zhi Piao, Kazuhiro Nakaya, and Akira Ito. "Molecular Characterization and Diagnostic Value ofTaenia solium Low-Molecular-Weight Antigen Genes." Journal of Clinical Microbiology 38, no. 12 (2000): 4439–44. http://dx.doi.org/10.1128/jcm.38.12.4439-4444.2000.

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Neurocysticercosis (NCC) caused by infection with the larvae ofTaenia solium is an important cause of neurological disease worldwide. In order to establish an enzyme-linked immunosorbent assay (ELISA) for this infection using recombinant proteins, we carried out molecular cloning and identified four candidates as diagnostic antigens (designated Ag1, Ag1V1, Ag2, and Ag2V1). Except for Ag2V1, these clones could encode a 7-kDa polypeptide, and Ag2V1 could encode a 10-kDa polypeptide. All of the clones were very similar. Except for Ag2V1, recombinant proteins were successfully expressed using anEscherichia coli expression system. Immunoblot analysis of NCC patient sera detected recombinant proteins, but because reactivity to recombinant Ag1 was too weak, Ag1 was not suitable as an immunodiagnostic antigen. So, Ag1V1 and Ag2 were chosen as ELISA antigens, and the Ag1V1/Ag2 chimeric protein was expressed. Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA. No assays of serum samples from patients with other parasitic infections recognized the Ag1V1/Ag2 chimeric protein. The Ag1V1/Ag2 chimeric protein obtained in this study had a high value for differential immunodiagnosis.

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Sproles, Ashley E., Anthony Berndt, Francis J. Fields, and Stephen P. Mayfield. "Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins." Applied Microbiology and Biotechnology 106, no. 4 (February 2022): 1677–89. http://dx.doi.org/10.1007/s00253-022-11790-9.

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Abstract The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products. Key points • A protein expression vector using double-antibiotic resistance genes was designed • Double antibiotic selection causes fewer colonies with more positive for phenotype • Coupling the new vector with FACS improves microalgal screening efficiency > 60%

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50

Rathore, Jitendra Singh, and Lalit Kumar Gautam. "Expression, Purification, and Functional Analysis of Novel RelE Operon fromX. nematophila." Scientific World Journal 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/428159.

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Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.Escherichia coliRelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin fromXenorhabdus nematophilapossessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter inE. coliTop 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

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