Co-expressions Of Protein Tyrosine Kinases EGFR-2, PDGFRβ And C-Src With Protein Tyrosine Phosphatase1B In Pichia Pastoris

Mediated by the balanced action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), the appropriate control of protein tyrosine phosphorylation is essential for cellular homeostasis. A strategy of co-expressing PTKs with PTPs was developed to overcome the apparent toxicity associated with expressing PTKs, and to allow the expression and preparation of active PTKs from P. pastoris. To test the hypothesis that the difficulty of expressing PTKs in P. pastoris was due to PTKs phosphorylating P. pastoris proteins, thus resulting in P. pastoris toxicity, we tryed to introduce a protein tyrosine phosphatase activity to overcome this apparent kinase activity-triggered problem. The gene encoding recombinant PTP1B was cloned into vector pAG32with blue fluorescent protein (BFP) and peroxisomal targeting signal1(SKL) to the C-terminus of the PTP1B, and it was cloned into vector pAG32with the four vectors pAG32S-PTP1B, pAG32S-PTP1B-SKL, PAG32S-BFP-PTP1B and pAG32S-BFP-PTP1B-SKLP to form the expression vectors by strain GS115in Pichia pastoris under the control of inducible AOX1promoter.The tyrosine kinases EGFR-2, PDGFRβ and c-Src are important with signal-transducing kinase activity. They are the most important therapeutic target for the treatment of cancer because of their crucial role in angiogenesis, which is fundamental to the malignancy of tumors. The tyrosine kinase EGFR-2, PDGFRβ or c-Src genes was cloned and expressed in vector pPIC3.5K with fused with His-tag and green fluorescent protein (GFP) at its N-terminus, in Pichia pastoris with PTP1B gene and the control GS115strain to get15strains, under the control of inducible AOX1promoter. To avoid the potential negative effect to the host cell, we added SKLto the C-terminus of the EGFR-2and PDGFRp and the resultant vectors were pPIC3.5-GFP-EGFR2-SKL, pPIC3.5-GFP-PDGFRβ-SKL.The right P. pastoris transformants were screened on his-deficient plates and YPD-G418plates by turns after electroporation of strains GS115with and without of PTP1B, and high yield strains were selected. The EGFR-2, PDGFRβ and c-Src fusion proteins were purified by Ni2+Sepharose. The activity of tyrosine kinases co-expressed with the PTP1B was compared to that produced without PTP1B in P. pastoris after grown in medium containing methanol. Fusion proteins of EGFR-2, PDGFRβ and c-Src were proved to be93kDa,89kDa, and92kDa, respectively, by western blot, and their tyrosine kinase activity were measured by ELISA. In the recombinant P. pastpris, compared with expressing tyrosine kinases alone, the tyrosine kinases co-expressed with PTPIB exhibited high kinase catalytic activity, the highest kinase catalytic activities were achieved by targeting the tyrosine kinases and PTPIB into peroxisomes. Further results showed that the EGFR-2, PDGFRβ and c-Src fusion proteins expressed in P. pastoris could be used as an attractive target for future anti-cancer therapeutics.