In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

Abstract Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

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A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

Plant Physiology ◽

10.1093/plphys/kiab471 ◽

2021 ◽

Author(s):

Adeline Harant ◽

Hsuan Pai ◽

Toshiyuki Sakai ◽

Sophien Kamoun ◽

Hiroaki Adachi

Keyword(s):

Nicotiana Benthamiana ◽

Cell Biology ◽

Plant Immunity ◽

Transient Gene Expression ◽

Coiled Coil ◽

Experimental System ◽

Vector System ◽

In Planta ◽

Functional Studies

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

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Tat-SF1 Protein Associates with RAP30 and Human SPT5 Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.5960 ◽

1999 ◽

Vol 19 (9) ◽

pp. 5960-5968 ◽

Cited By ~ 36

Author(s):

Jae B. Kim ◽

Yuki Yamaguchi ◽

Tadashi Wada ◽

Hiroshi Handa ◽

Phillip A. Sharp

Keyword(s):

Recombinant Proteins ◽

Transcriptional Activity ◽

Transcription Activation ◽

Cellular Proteins ◽

Pol Ii ◽

Nuclear Extracts ◽

Cellular Factors ◽

Immunodeficiency Virus

ABSTRACT The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.

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The Valine Anticodon and Valylatability of Peanut Clump Virus RNAs Are Not Essential but Provide a Modest Competitive Advantage in Plants

Journal of Virology ◽

10.1128/jvi.74.18.8720-8725.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8720-8725 ◽

Cited By ~ 8

Author(s):

Daiki Matsuda ◽

Patrice Dunoyer ◽

Odile Hemmer ◽

Christiane Fritsch ◽

Theo W. Dreher

Keyword(s):

Sequence Analysis ◽

Nicotiana Benthamiana ◽

General Characteristic ◽

Wild Type ◽

Genomic Rnas ◽

Viral Rnas ◽

Fold Reduction ◽

Direct Competition

ABSTRACT The role of valine aminoacylation of the two genomic RNAs ofPeanut clump virus (PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GΔC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3′ end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GΔC anticodon that does not support valylation. The presence in RNA1 of GΔC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GΔC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in Nicotiana benthamiana plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in N. benthamiana plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV.

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Different expression levels of two KgmB-His fusion proteins

Journal of the Serbian Chemical Society ◽

10.2298/jsc0512401m ◽

2005 ◽

Vol 70 (12) ◽

pp. 1401-1407 ◽

Cited By ~ 3

Author(s):

Sandra Markovic ◽

Sandra Vojnovic ◽

Milija Jovanovic ◽

Branka Vasiljevic

Keyword(s):

Recombinant Proteins ◽

Fusion Proteins ◽

Kanamycin Resistance ◽

Expression Vectors ◽

E Coli ◽

C Terminus ◽

N Terminus ◽

Different Levels ◽

Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

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A Brief History of the Discovery of Amelogenin Nanoribbons In Vitro and In Vivo

Journal of Dental Research ◽

10.1177/00220345211043463 ◽

2021 ◽

pp. 002203452110434

Author(s):

Y. Bai ◽

J. Bonde ◽

K.M.M. Carneiro ◽

Y. Zhang ◽

W. Li ◽

...

Keyword(s):

Recombinant Proteins ◽

Protein Structures ◽

Historical Review ◽

Limited Information ◽

Transmission Electron Microscopy Analysis ◽

Electron Microscopy Analysis ◽

History Of ◽

The 1960S

Without evidence for an organic framework, biological and biochemical processes observed during amelogenesis provided limited information on how extracellular matrix proteins control the development of the complex fibrous architecture of human enamel. Over a decade ago, amelogenin nanoribbons were first observed from recombinant proteins during in vitro mineralization experiments in our laboratory. In enamel from mice lacking the enzyme kallikrein 4 (KLK4), we later uncovered ribbon-like protein structures that matched the morphology, width, and thickness of the nanoribbons assembled by recombinant proteins. Interestingly, similar structures had already been described since the 1960s, when enamel sections from various mammals were demineralized and stained for transmission electron microscopy analysis. However, at that time, researchers were not aware of the ability of amelogenin to form nanoribbons and instead associated the filamentous nanostructures with possible imprints of mineral ribbons in the gel-like matrix of developing enamel. Further evidence for the significance of amelogenin nanoribbons for enamel development was stipulated when recent mineralization experiments succeeded in templating and orienting the growth of apatite ribbons along the protein nanoribbon framework. This article provides a brief historical review of the discovery of amelogenin nanoribbons in our laboratory in the context of reports by others on similar structures in the developing enamel matrix.

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mRNA Engineering for the Efficient Chaperone-Mediated Co-Translational Folding of Recombinant Proteins in Escherichia coli

International Journal of Molecular Sciences ◽

10.3390/ijms20133163 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3163 ◽

Cited By ~ 1

Author(s):

Le Minh Bui ◽

Almando Geraldi ◽

Thi Thuy Nguyen ◽

Jun Hyoung Lee ◽

Ju Young Lee ◽

...

Keyword(s):

Recombinant Proteins ◽

Molecular Chaperones ◽

Rna Binding ◽

Protein Translation ◽

Soluble Form ◽

Mrna Transcript ◽

Protein Substrates ◽

Limited Success ◽

Intergenic Sequences

The production of soluble, functional recombinant proteins by engineered bacterial hosts is challenging. Natural molecular chaperone systems have been used to solubilize various recombinant proteins with limited success. Here, we attempted to facilitate chaperone-mediated folding by directing the molecular chaperones to their protein substrates before the co-translational folding process completed. To achieve this, we either anchored the bacterial chaperone DnaJ to the 3ʹ untranslated region of a target mRNA by fusing with an RNA-binding domain in the chaperone-recruiting mRNA scaffold (CRAS) system, or coupled the expression of DnaJ and a target recombinant protein using the overlapping stop-start codons 5ʹ-TAATG-3ʹ between the two genes in a chaperone-substrate co-localized expression (CLEX) system. By engineering the untranslated and intergenic sequences of the mRNA transcript, bacterial molecular chaperones are spatially constrained to the location of protein translation, expressing selected aggregation-prone proteins in their functionally active, soluble form. Our mRNA engineering methods surpassed the in-vivo solubilization efficiency of the simple DnaJ chaperone co-overexpression method, thus providing more effective tools for producing soluble therapeutic proteins and enzymes.

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Overexpression of Trigger Factor Prevents Aggregation of Recombinant Proteins in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.66.3.884-889.2000 ◽

2000 ◽

Vol 66 (3) ◽

pp. 884-889 ◽

Cited By ~ 186

Author(s):

Kazuyo Nishihara ◽

Masaaki Kanemori ◽

Hideki Yanagi ◽

Takashi Yura

Keyword(s):

Escherichia Coli ◽

Recombinant Proteins ◽

Protein Production ◽

Trigger Factor ◽

Human Lysozyme ◽

E Coli ◽

Constructed Plasmids ◽

Expression Plasmids

ABSTRACT To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli, we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production inE. coli.

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CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins

Planta ◽

10.1007/s00425-019-03180-9 ◽

2019 ◽

Vol 250 (2) ◽

pp. 463-473 ◽

Cited By ~ 2

Author(s):

Kouki Matsuo ◽

Go Atsumi

Keyword(s):

Transient Expression ◽

Recombinant Proteins ◽

Nicotiana Benthamiana

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DNA Vaccination by Electroporation and Boosting with Recombinant Proteins Enhances the Efficacy of DNA Vaccines for Schistosomiasis Japonica

Clinical and Vaccine Immunology ◽

10.1128/cvi.00231-09 ◽

2009 ◽

Vol 16 (12) ◽

pp. 1796-1803 ◽

Cited By ~ 27

Author(s):

Yang Dai ◽

Yinchang Zhu ◽

Donald A. Harn ◽

Xiaoting Wang ◽

Jianxia Tang ◽

...

Keyword(s):

Adult Worm ◽

Recombinant Proteins ◽

Vaccine Efficacy ◽

Dna Vaccines ◽

Interleukin 2 ◽

Schistosomiasis Japonica ◽

Protein Vaccine ◽

Control Programs ◽

Protein Vaccines

ABSTRACT Schistosomiasis japonica is an endemic, zoonotic disease of major public health importance in China. Control programs combining chemotherapy and snail killing have not been able to block transmission of infection in lakes and marsh regions. Vaccination is needed as a complementary approach to the ongoing control programs. In the present study, we wanted to determine if the efficacies of DNA vaccines encoding the 23-kDa tetraspanin membrane protein (SjC23), triose phosphate isomerase (SjCTPI), and sixfold-repeated genes of the complementarity determining region 3 (CDR3) in the H chain of NP30 could be enhanced by boosting via electroporation in vivo and/or with cocktail protein vaccines. Mice vaccinated with cocktail DNA vaccines showed a significant worm reduction of 32.88% (P < 0.01) and egg reduction of 36.20% (P < 0.01). Vaccine efficacy was enhanced when animals were boosted with cocktail protein vaccines; adult worm and liver egg burdens were reduced 45.35% and 48.54%, respectively. Nearly identical results were obtained in mice boosted by electroporation in vivo, with adult worm and egg burdens reduced by 45.00% and 50.88%, respectively. The addition of a protein vaccine boost to this regimen further elevated efficacy to approximately 60% for adult worm burden and greater than 60% for liver egg reduction. The levels of interleukin-2, gamma interferon, and the ratios of immunoglobulin G2a (IgG2a)/IgG1 clearly showed that cocktail DNA vaccines induced CD4+ Th1-type responses. Boosting via either electroporation or with recombinant proteins significantly increased associated immune responses over those seen in mice vaccinated solely with DNA vaccines. Thus, schistosome DNA vaccine efficacy was significantly enhanced via boosting by electroporation in vivo and/or cocktail protein vaccines.

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