title: Co-expression of protein tyrosine kinases EGFR-2 and PDGFRβ with protein tyrosine phosphatase 1B in Pichia pastoris.
abstract: 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 Ni(2+) 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, P. R. China.
authors.forename: Pham Ngoc
authors.initials: PN
authors.lastname: Tu
authors.forename: Yamin
authors.initials: Y
authors.lastname: Wang
authors.forename: Menghao
authors.initials: M
authors.lastname: Cai
authors.forename: Xiangshan
authors.initials: X
authors.lastname: Zhou
authors.forename: Yuanxing
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular; D015870:Gene Expression; D060847:Metabolic Engineering / Q000379:methods; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
chemical_list: D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D020797:Receptor, Platelet-Derived Growth Factor beta; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1