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KMID : 0545120140240020152
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Journal of Microbiology and Biotechnology
2014 Volume.24 No. 2 p.152 ~ p.159
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Co-Expression of Protein Tyrosine Kinases EGFR-2 and PDGFRβ with Protein Tyrosine Phosphatase 1B in Pichia pastoris
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Pham Ngoc Tu
Yamin Wang
Menghao Cai
Xiangshan Zhou
Yuanxing Zhang
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Abstract
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The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni2+ affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics.
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KEYWORD
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Protein tyrosine kinase, Protein tyrosine phosphatase, Peroxisome, Pichia pastoris
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