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| Expression and Purification of Functional HuGALNT3 without the Transmembrane Domain (huGALNT3-sol) in Pichia pastoris |
| KONG Yun1, GAO Hai-tao1, LI Shu-fang1, WANG Peng1,2, GU Guo-feng2, GU Li1,2 |
1. State key laboratory for Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China;
2. National Glycoengineering Research Center, Jinan 250100, China |
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Abstract Objective:In order to research the bioactivity of GALNT3, the truncated part of GALNT3 (huGALNT3-sol) which was deleted of the hydrophobic trans-membrane domain were obtained using Pichia pastoris expression system, and assayed the transferring GalNAc activity of recombinant huGALNT3-sol. Methods: The gene of human GALNT3-sol (1 755 bp)was amplified from pET15b/ GALNT3-sol and cloned into expression vector pPIC9k, and the recombinant plasmid was transformed into Pichia pastoris GS115 strain through electroporation. The high copy recombinant strains with high-level huGALNT3-sol production were screened out by MD plate and G418. High level of huGALNT3-sol was obtained in BMMY medium the induction of methanol, and purified from the supernatant with Ni-NAT.The identity of the recombinant protein was confirmded by SDS-PAGE and then Western blotting analysis. HPLC and MALDI-TOF-MS analysis were used to identify the bioactivity of recombinant huGALNT3-sol. Results:The recombinant Pichia pastoris which could secretory express the human GALNT3-sol protein was constructed successfully. High level of huGALNT3-sol was obtained in BMMY medium (pH 6.0) after 96 hours induction of 20℃ and 0.5% methanol, with the highest yield of 5mg/L in shake flask culture. The identity of the recombinant protein was confirmed by Western blot analysis and the huGALNT3-sol expressed in Pichia pastoris is in higher molecular weight glycoforms. The activity assay showed the recombinant huGALNT3-sol has the activity of transferring GalNAc to Ser/Thr residues in peptide. Conclusion:The human GALNT3-sol, which has the activity of transferring GalNAc to Ser/Thr residues in peptide, was successfully expressed and purified in Pichia pastoris. It provides support for the further research and application of GALNT3.
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Received: 13 January 2012
Published: 25 May 2012
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