Abstract:
:A stably transformed insect cell expression system is superior to a baculovirus expression system, since the expression system is sustained and there is no cell lysis, but the isolation of cell lines producing recombinant proteins is time-consuming and laborious. In this study, we developed a technique for the rapid isolation and efficient cultivation of sorted cells in a 24 well deep plate Bioshaker, utilizing the fluorescence activated cell-sorting (FACS) method. TnpXme11 cells, which stably expressed GFP(uv)-beta1,3-N-acetylglucosaminyltransferase2 (GGT2), were transfected with a plasmid vector for the expression of a molecular chaperone (TnpXme11-hCNX6 cell line). The expression levels of GGT2 and the molecular chaperone fused with HcRed were analyzed by FACS. Two cell lines were established by single and double sorting. Sorting of the top 10% of the TnpXme11-hCNX6 cell population with the highest fluorescence yielded the TnpXme11-hCNX6-1 cell line. TnpXme11-hCNX6-2 cells were created in a similar fashion, as mentioned above, by a second sorting of the top 10% of the TnpXme11-hCNX6-1 cell population with the highest fluorescence. The cells thus isolated produced approximately 2-fold higher extracellular activity than that before cell sorting. This procedure can be accomplished in only 2 weeks, including transfection, isolation and analysis of high protein-producing cells, and is a breakthrough strategy for the rapid isolation of a recombinant, stable insect cell line.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Kato T,Yoshizuka K,Park EYdoi
10.1016/j.jbiotec.2010.03.011subject
Has Abstractpub_date
2010-05-17 00:00:00pages
102-7issue
2eissn
0168-1656issn
1873-4863pii
S0168-1656(10)00156-2journal_volume
147pub_type
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