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. 2004 Nov 1;10(21):3188-90.
doi: 10.3748/wjg.v10.i21.3188.

Expression of Human Augmenter of Liver Regeneration in Pichia Pastoris Yeast and Its Bioactivity in Vitro

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Free PMC article

Expression of Human Augmenter of Liver Regeneration in Pichia Pastoris Yeast and Its Bioactivity in Vitro

Qi Liu et al. World J Gastroenterol. .
Free PMC article

Abstract

Aim: To construct a yeast expression system of human augmenter of liver regeneration (hALR) and to examine its bioactivity in vitro.

Methods: With PCR and gene recombination techniques, cDNA of open reading frame of hALR was obtained from recombinant plasmid pcDNA3.1-hALR and inserted into plasmid pPIC9. The cDNA of hALR from recombinant plasmid pPIC9-hALR demonstrated by sequencing was subcloned into plasmid pPIC9K. The recombinant plasmid pPIC9K-hALR was transformed into GS115 with electroporation. hALR was expressed by GS115 under the induction of 5 mL/L methanol and purified with ultrafiltration after it was analyzed by 15% SDS-PAGE and Western blot. The effects of hALR on in vitro proliferation of QGY and HepG(2) cells were evaluated by (3)H-TdR methods.

Results: The correctness and integrity of recombinant plasmids pPIC9-hALR and pPIC9K-hALR were identified by restriction digestion, PCR and sequencing methods, respectively. hALR as a secretive protein was successfully expressed by GS115. Its molecular weight was about 15 ku and the target protein was about 60% of the total protein in the supernatant from GS115 with plasmid pPIC9K-hALR. The results of Western blot of hALR showed the specific band. The high qualitative hALR was obtained through ultrafiltration. hALR could stimulate in vitro proliferation of QGY and HepG(2) cells in a dose-dependent manner, but there was a difference in reactivity to hALR between QGY and HepG(2).

Conclusion: The hALR as a secretive protein can be successfully expressed by GS115. It may stimulate in vitro proliferation of QGY and HepG(2) cells at a dose-dependent manner. But QGY and HepG(2) cells have different reactivities to hALR.

Figures

Figure 1
Figure 1
Identifications of recombination plasmid. Lane 1: Am-plified from pPIC9K; Lane 2: Amplified from pPIC9K-hALR; Lane 3: pPIC9-hALR digested by XhoI + NotI; Lane M: 1 kb DNA ladder; Lane 4: pPIC9K-hALR digested by SacI + SalI (6.3 kb + 3.4 kb); Lane 5: 3.4 kb fragment with hALR.
Figure 2
Figure 2
SDS-PAGE analysis of purified hALR. Lane M: Pro-tein marker; Lanes 1, 2, and 3: Purified hALR.
Figure 3
Figure 3
Western blot analysis of supernatants from GS115 treated with different plasmids. Lane 1: Supernatant from GS115 with pPIC9K; Lane 2: Supernatant from GS115 with pPIC9K- hALR.

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