One limitation of baculovirus expression system is its inability to produce recombinant glycoprotein containing authentic N-glycan (authentic refers to mammalian N-glycan). After years of hard work of researchers, this limitation had been addressed by constitutively expressing the enzyme coding genes required for mammalian glycosylation under the control of the promoter of insect cells . However, the metabolic load resulted from simultaneous expression of multiple gene may compromise the stability of the transgenic cell lines.
Although the advantages of the glycoprotein expression pathway via gene modified insect cell lines are still obvious, however, as a expert in the field of protein expression, Creative BioMart had developed a N-Linked Glycoprotein Expression Service based on a more advanced cell line—SfSWT-5, which is a combination of glycoengineering and inducible mammalian-like protein N-glycosylation machinery.
Instead of continuously express, the inserted glycosylation related enzymes can only be expressed under the induction of certain substrates or baculovirus. It can significantly alleviate the metabolic load of cells and consequently increase the stability.
A gene package that contains 6 mammalian glycosylation related enzymes coding gene is combined with plasmids encoding a tetracycline-responsive transactivator and the selectable marker and then transformed into the Sf9 cell line. The isolated transgenic Sf9 cell derivative is then named as SfSWT-5. As the gene package is placed under the tetracycline-responsive transactivator, the 6 content enzymes will not be transcribe unless induced by doxycycline. Also, baculovirus infection can also stimulate the promoter to perform strong expression.
Note, the 6 enzymes refer to human β1,2-N-acetylglucosaminyltransferase II (GnTII), bovine β1,4-galactosyltransferase (β4GalTI), rat α2,6-sialyltransferase (ST6GalI), murine α2,3-sialyltransferase III (ST3GalIII) murine sialic acid synthase (SAS) and murine cytosine-5'- monophospho-sialic acid synthetase (CSAS).
In the absence of doxycycline, only low level of ST6GalI and ST3GalIII is detected by the RT-PCR. On the contrary, all the 6 enzymes are detectable in a high level if doxycycline is added into the medium (Figure 1).
Figure. 1. RT–PCR assay of induction of transgene expression in Sf9 and SfSWT-5 cells. Incubated for 48 h with (+) or without (−)doxycycline with (RT+) or without (RT−) reverse transcriptase
Both viral infection and doxycycline can induce transcription. However, when the cells were infected with baculovirus and cultured in the presence of doxycycline, the induction effect was stronger
Figure 2. The glycoengineered phenotype of low- and high-passage SfSWT-5 cells. Incubated for 24 h and then mock-infected (A) or infected (B) with a wild-type baculovirus.
The results showed that there were no obvious, short-term and adverse changes in the growth characteristics of SfSWT-5 cells during the expression of the target protein. After 300 passages, the growth characteristics of the cells remained unchanged.
Compared with the parental Sf9 cell line, either the number of passages (Figure 2) nor the introduction of 6 transgenes (Figure 3) will not reduce the glycoprotein production capacity of SfSWT-5 cells.
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