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제안 방법
- Accordingly, this study explored the expression of the truncated-mutant t-PA in CHO DG44 cells when combining features from transient and stable transfection [5] in an orbitally shaking disposable bioreactor (TubeSpin, TPP Trasadingen, Switzerland), plus a rapid approach for producing the truncated-mutant t-PA is developed.
- 2/blunt Cloning Vector, using a CloneJET PCR Cloning Kit from Fermentas (Lithuania) based on the manufacturer’s procedures. After confirming the proper sequence arrangement by bidirectional sequencing, two upstream and downstream BglII restriction sites of pJET were used for cloning into the EcoRV site in pTracer-SV40 (CHO expression vector). The BglII sticky ends were converted to blunt ends using the CloneJET PCR Cloning Kit DNA blunting enzyme.
- As a result, a novel truncated form of t-PA with an improved fibrin affinity was expressed in a CHO DG44 expression system [9]. As a further improvement, a PAI-1-resistant novel form of the truncated t-PA was also designed. The truncated mutant variant was then successfully expressed in CHO DG44 and showed an increased resistance to PAI-1 [8].
- The clones were tested for their expression of the t-PA protein. Positive clones were expanded into large wells and then into flasks or plates, and re-tested to confirm their expression.
- SDS-PAGE (12%) was carried out under reduced conditions and the Coomassie Blue-stained gel scanned using a densitometer (Bio-Rad) to determine the expression level of the truncated mutant t-PA in the culture supernatant.
- The CHO DG44 cells were stably transfected with the truncated mutant t-PA-p-Tracer-SV40 recombinant plasmid based on the optimized Lipofectamine:DNA ratio mentioned above, and the supernatant was harvested on different days (day 3, day 6, day 9, and day 11) after the transfection. The expression of the fusion protein on day 7 was analyzed by SDS-PAGE.
- The supernatant from the static and suspension cultures was sampled on different days throughout the experiment to assess the protein production kinetics. An ELISA-based biofunctional immunosorbent assay with a Biopool Chromolize t-PA assay kit was used to quantify the truncated-mutant t-PA.
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