Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

Andersen, Søren

doi:10.1251/bpo78

Cited by 8 publications

(9 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, in the insect cells, the glycosylation pattern of recombinant glycoproteins has not been characterized under various conditions such as cell type, postinfection (PI) time, and sampling location. The predominantly used cell lines for the baculovirus expression system include the Spodoptera frugiperda ‐derived cell line “Sf9” and the Trichoplusia ni ‐derived cell line “High Five.” The High Five cell line is used more often than the Sf9 cell line because it replicates rapidly and secretes recombinant glycoproteins at a higher rate than the Sf9 cell line . Thus, the harvesting time after the virus infects the cells (PI time) directly and affects the recombinant protein expression as well as glycosylation.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Glycan Structures of GlycoproteinGA733‐Fc Expressed in a Baculovirus‐Insect Cell System

Lee

2015

Bulletin Korean Chem Soc

View full text Add to dashboard Cite

Baculovirus-insect cell systems have been used to express functional recombinant biopharmaceutical proteins. Two pFastBac Dual vectors carrying the gene encoding antigen GA733, a cell-surface glycoprotein, fused to the IgG Fc (GA733-Fc) or KDEL (endoplasmic reticulum [ER] retention sequence) (GA733-FcK) genes were constructed to generate baculoviruses expressing the corresponding recombinant proteins in insect cells. The expression of the recombinant GA733-Fc and GA733-FcK proteins and their glycan structure profiles were assessed under various conditions by analyzing the cell line (Sf9 and High Five), the postinfection (PI) time (48, 72, and 96 h), and the harvested sample (cell culture media [CM] or cell lysate [CL]). Immunoblotting showed nonidentical expression of both GA733-Fc and GA733-FcK under various conditions. Glycosylation analysis revealed that varying conditions affected the glycan structure profiles. These results suggest that the PI time, subcellular localization, and cell type affect recombinant protein expression as well as glycosylation in the baculovirus-insect cell system. ExperimentalBacmid Generation. The GA733-Fc and GA733-FcK genes were cloned under the control of the polyhedrin promoter

show abstract

Section: Introductionmentioning

confidence: 99%

Characterization of the Glycan Structures of GlycoproteinGA733‐Fc Expressed in a Baculovirus‐Insect Cell System

Lee

2015

Bulletin Korean Chem Soc

View full text Add to dashboard Cite

show abstract

“…The High Five cell line is used more widely that the Sf9 cell line because it produces and secretes proteins at a higher rate than the Sf9 cell line (Wickham et al . ; Vallazza & Petri ; Andersen ). The time at which a sample is harvested after virus infection (post‐infection time) is also an important factor that affects recombinant protein expression.…”

Section: Introductionmentioning

confidence: 99%

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

Kim

Lee

et al. 2014

Entomological Research

View full text Add to dashboard Cite

The baculovirus-insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell-surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733-Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post-infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733-Fc gene were constructed to express GA733-Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733-Fc at 96 h post-infection at 0.1 MOI and with GA733-FcK at 96 h post-infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733-Fc at 48 h post-infection at 1 MOI and with GA733-FcK at 48 h post-infection at 3 MOI, respectively. These results suggest that the MOI value, post-infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus-insect cell system.

show abstract

“…Thus, glutamate transporters in the synaptic vesicle membrane play an important role in directing glutamate to the neurotransmitter pathway away from the metabolic pathway. Three vesicular glutamate transporters (VGLUT1–3) have recently been identified in neuronal tissue where they play a key role in maintaining the vesicular glutamate level (Ni et al, 1994; Herzog et al, 2001, 2004; Andersen, 2004). VGLUT1 and VGLUT2 are the predominant isoforms (Fremeau et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Vesicular glutamate transporters VGLUT1 and VGLUT2 in the developing mouse barrel cortex

Liguz-Lecznar

Skangiel-Kramska

2007

Intl J of Devlp Neuroscience

View full text Add to dashboard Cite

Three vesicular glutamate transporters have been identified in mammals. Two of them, VGLUT1 and VGLUT2, define the glutamatergic phenotype and their distribution in the brain is almost complementary. In the present study we examined the distribution and expression levels of these two VGLUTs during postnatal development of the mouse barrel cortex. We also investigated changes in the localization of VGLUT1 and VGLUT2 within particular compartments of the barrel field (barrels/septa) during its development. We found differences in the time course of developmental expression, with VGLUT1 peaking around P14, while VGLUT2 increased gradually until adulthood. Over the examined period (P3 - adult) both transporters had stronger expression in the barrel interiors, and in this compartment VGLUT2 dominated, whereas in the inter-barrel septa VGLUT1 dominated over VGLUT2. Furthermore, we found that some nerve terminals in the barrel cortex coexpressed both transporters until adulthood. Colocalization was observed within the barrels, but not within the septa.

show abstract

Expression and purification of recombinant vesicular glutamate transporter VGLUT1 using PC12 cells and High Five insect cells

Cited by 8 publications

References 21 publications

Characterization of the Glycan Structures of GlycoproteinGA733‐Fc Expressed in a Baculovirus‐Insect Cell System

Characterization of the Glycan Structures of GlycoproteinGA733‐Fc Expressed in a Baculovirus‐Insect Cell System

Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

Vesicular glutamate transporters VGLUT1 and VGLUT2 in the developing mouse barrel cortex

Contact Info

Product

Resources

About