scholarly journals Secreted Trimeric Chikungunya Virus Spikes from Insect Cells: Production, Purification, and Glycosylation Status

Processes ◽  
2022 ◽  
Vol 10 (1) ◽  
pp. 162
Author(s):  
Tessy A. H. Hick ◽  
Corinne Geertsema ◽  
Maurice G. L. Henquet ◽  
Dirk E. Martens ◽  
Stefan W. Metz ◽  
...  

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and <2% have complex-type N-glycans. The LC –MS/MS analysis also revealed three O-glycosylation sites in E1. In conclusion, the trimeric, glycosylated CHIKV spikes have been successfully produced in insect cells and are now available for vaccination studies.

1992 ◽  
Vol 286 (3) ◽  
pp. 677-680 ◽  
Author(s):  
J D Robishaw ◽  
V K Kalman ◽  
K L Proulx

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.


F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 82 ◽  
Author(s):  
Lyle R. Petersen ◽  
Ann M. Powers

Chikungunya virus is a mosquito-borne alphavirus that causes fever and debilitating joint pains in humans. Joint pains may last months or years. It is vectored primarily by the tropical and sub-tropical mosquito, Aedes aegypti, but is also found to be transmitted by Aedes albopictus, a mosquito species that can also be found in more temperate climates. In recent years, the virus has risen from relative obscurity to become a global public health menace affecting millions of persons throughout the tropical and sub-tropical world and, as such, has also become a frequent cause of travel-associated febrile illness. In this review, we discuss our current understanding of the biological and sociological underpinnings of its emergence and its future global outlook.


1992 ◽  
Vol 286 (3) ◽  
pp. 819-824 ◽  
Author(s):  
K Rose ◽  
G Turcatti ◽  
P Graber ◽  
S Pochon ◽  
P O Regamey ◽  
...  

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.


1995 ◽  
Vol 17 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Miguel Medina ◽  
Abelardo L�pez-Rivas ◽  
Douwe Zuidema ◽  
Graham J. Belsham ◽  
Esteban Domingo ◽  
...  

1995 ◽  
Vol 312 (3) ◽  
pp. 847-853 ◽  
Author(s):  
M Tomita ◽  
N Ohkura ◽  
M Ito ◽  
T Kato ◽  
P M Royce ◽  
...  

We have investigated the expression of human procollagen III by insect cells infected with a recombinant baculovirus carrying cDNA for the pro-alpha1(III) chain of type-III collagen. A high level of expression was obtained, and a small proportion of the heterologously expressed pro-alpha1(III) chains formed normally disulphide-bonded procollagen III, which was secreted into the culture medium. This species displayed a melting temperature (Tm) of approx. 38 degrees C as assessed by its resistance to digestion by a mixture of trypsin and chymotrypsin, slightly lower than that of 39.5 degrees C for procollagen III synthesized by cultured human dermal fibroblasts, and reflected a slight degree of under-hydroxylation of prolyl residues. This is possibly a consequence of the lower incubation temperature of insect cells, or of an insufficiency of prolyl hydroxylase activity within them. A significant proportion of the expressed chains formed trimeric molecules of similar thermal stability containing an apparently full-length triple-helical region, but were not disulphide-bonded and not secreted. In addition to providing a source of recombinant human procollagen III, the system promises to be useful in the study of procollagen chain association and subsequent folding.


2020 ◽  
Vol 38 (2) ◽  
pp. 153 ◽  
Author(s):  
Sim Sai ◽  
Viroj Wiwanitkit

Coronavirus disease 2019 (COVID-19) is a new viral disease that has become a global public health concern in 2020. This disease is caused by a novel coronavirus firstly detected in the People’s Republic of China. This viral infection can cause febrile illness and respiratory problems. There are other uncommon clinical presentations of COVID-19 such as afebrile illness, diarrhea, non-coughing and confusion. These atypical presentations can result in difficulty in diagnosing the disease. In this short review, the authors specifically discuss the important uncommon atypical presentations of COVID-19.


1991 ◽  
Vol 59 (5) ◽  
pp. 1627-1632 ◽  
Author(s):  
I G Charles ◽  
B C Rodgers ◽  
A J Makoff ◽  
S N Chatfield ◽  
D E Slater ◽  
...  

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