GFP-Based Expression Screening of Membrane Proteins in Insect Cells Using the Baculovirus System

2014 ◽  
pp. 197-209 ◽  
Author(s):  
Nien-Jen Hu ◽  
Heather Rada ◽  
Nahid Rahman ◽  
Joanne E. Nettleship ◽  
Louise Bird ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Insect Cells ◽  
Expression Screening ◽  
Baculovirus System

scholarly journals Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis

2008 ◽  
Vol 8 (1) ◽  
pp. 51 ◽  
Author(s):  
Huajun Qin ◽  
Jian Hu ◽  
Yuanzhi Hua ◽  
Shridhar V Challa ◽  
Timothy A Cross ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Membrane Proteins ◽  
High Throughput ◽  
Cloning And Expression ◽  
Expression Screening

Functional overexpression and characterization of human bradykinin subtype 2 receptor in insect cells using the baculovirus system

2006 ◽  
Vol 99 (3) ◽  
pp. 868-877 ◽  
Author(s):  
Arun Kumar Shukla ◽  
Winfried Haase ◽  
Christoph Reinhart ◽  
Hartmut Michel
Keyword(s):  
Insect Cells ◽  
Baculovirus System

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

2010 ◽  
Vol 149 (2) ◽  
pp. 219-227 ◽  
Author(s):  
A. Usami ◽  
S. Ishiyama ◽  
C. Enomoto ◽  
H. Okazaki ◽  
K. Higuchi ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Insect Cells ◽  
Recombinant Protein Expression ◽  
Baculovirus System

Transient transfection coupled to baculovirus infection for rapid protein expression screening in insect cells

2012 ◽  
Vol 179 (1) ◽  
pp. 46-55 ◽  
Author(s):  
Stephan Radner ◽  
Patrick H.N. Celie ◽  
Karoline Fuchs ◽  
Werner Sieghart ◽  
Titia K. Sixma ◽  
...  
Keyword(s):  
Protein Expression ◽  
Insect Cells ◽  
Transient Transfection ◽  
Expression Screening ◽  
Baculovirus Infection

scholarly journals Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins

2020 ◽  
Author(s):  
Fei Jin ◽  
Yao Wang ◽  
Mengqi Wang ◽  
Minxuan Sun ◽  
Motoyuki Hattori
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Size Exclusion Chromatography ◽  
Fluorescence Detection ◽  
Size Exclusion ◽  
Expression Screening ◽  
Functional Studies ◽  
Membrane Protein Expression ◽  
Exclusion Chromatography

AbstractMembrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

The use of baculovirus vectors for the production of membrane proteins in insect cells

1999 ◽  
Vol 27 (6) ◽  
pp. 928-932 ◽  
Author(s):  
Robert D. Possee ◽  
Carole J. Thomas ◽  
Linda A. King
Keyword(s):  
Membrane Proteins ◽  
Insect Cells

scholarly journals Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

10.3791/52357 ◽  
2015 ◽  
Author(s):  
Louise E. Bird ◽  
Heather Rada ◽  
Anil Verma ◽  
Raphael Gasper ◽  
James Birch ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Expression Screening ◽  
Green Fluorescent

scholarly journals High-level expression of biologically active recombinant bovine follicle stimulating hormone in a baculovirus system

1998 ◽  
Vol 20 (1) ◽  
pp. 83-98 ◽  
Author(s):  
DF van de Wiel ◽  
PA van Rijn ◽  
RH Meloen ◽  
RJ Moormann
Keyword(s):  
Untranslated Region ◽  
Insect Cells ◽  
Biological Activities ◽  
Biologically Active ◽  
Beta Subunit ◽  
Significant Activity ◽  
Molecular Masses ◽  
Baculovirus System ◽  
High Level

Superovulation treatment of cows can benefit from the application of very pure recombinant bovine FSH (rbFSH), which is produced in nonmammalian cells. rbFSH is completely free of LH, and therefore can possibly reduce the variability in the results of superovulation. Furthermore, it does not contain brain-tissue-derived proteins and, when produced under serum-free conditions, it is free of other mammalian substances or potentially infectious material. We have produced rbFSH in insect cells, with the ultimate aim of inducing superovulation in cattle. Sf21 insect cells were coinfected with two recombinant baculoviruses, containing the cDNAs of bovine FSH alpha- and beta-subunits respectively. High levels of production of bioactive rbFSH were obtained after cloning cDNA that contained a major part of the 3' untranslated region of the bFSH beta gene. Maximum production of rbFSH 1-5 micrograms/ml (as measured by immunoassay) was obtained 70-90 h after infection. The recombinant material was highly potent in two in vitro bioassays, giving biological activities of 13 IU/ml (Y1 cell rounding assay), 22 IU/ml (Y1 cell cAMP assay), and 23 IU/ml (bovine oocyte maturation inhibition assay), and had a lower but significant activity of 6 IU/ml in the rat Sertoli cell assay. rbFSH was purified by immunoaffinity chromatography, using a monoclonal antibody directed against the human FSH beta-subunit. The purified heterodimer appeared to be homogeneous by SDS-PAGE, whereas the free beta-subunit appeared as a doublet, possibly indicating differently glycosylated forms. Intact heterodimer and both subunits were further identified by western blot analysis, and showed apparent molecular masses of 20 kDa (alpha-subunit), 23 kDa (beta-subunit) and 32.5 kDa (heterodimer). This insect-cell-produced rbFSH did not bind to wheat germ agglutinin, thus indicating that glycosidic side-chains may not contain terminal sialic acid. The relevance of a large 3' untranslated region in bFSH beta cDNA to the level of production of rbFSH, and the possible implications of the pattern of glycosylation for the biological activity of the recombinant hormone are discussed.

scholarly journals Expression screening of integral membrane proteins fromHelicobacter pylori26695

2007 ◽  
Vol 16 (12) ◽  
pp. 2667-2676 ◽  
Author(s):  
Georgios Psakis ◽  
Sandra Nitschkowski ◽  
Caterina Holz ◽  
Daniel Kreß ◽  
Manuel Maestre-Reyna ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Expression Screening
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