猪hnRNPK蛋白与酪氨酸蛋白激酶c-Src的互作分析

doi:10.11843/j.issn.0366-6964.2018.02.003

Abstract

Abstract:

This experiment was conducted to investigate the interaction between porcine heterogeneous nuclear ribonucleoprotein K (hnRNPK) protein and non-receptor tyrosine protein kinase (c-Src) containing SRC Homology (SH3) domain. The yeast two-hybrid expressing vectors pGBKT7-hnRNPK, pGADT7-c-Src, pGADT7-ΔSH3, pGADT7-ΔSH2, pGADT7-ΔPTKc and pGADT7-SH3 were constructed using PCR, enzyme digestion and ligation methods, and then transformed into the yeast strain AH109 by PEG-LiAc method, and self-activation activity of bait and prey proteins in yeast cells were tested. Furthermore, the interaction between porcine hnRNPK and different mutants of c-Src protein were analyzed using yeast two-hybrid assay in vivo. With the DNA recombinant technology, the plasmids pET28a-hnRNPK, pGEX-6p1-c-Src, pGEX-6p1-ΔSH3, pGEX-6p1-ΔSH2, pGEX-6p1-ΔPTKc and pGEX-6p1-SH3 were constructed. Then the plasmids were transformed into E. coli BL21 and induced to express the target proteins by IPTG. GST and His tagged fusion proteins were purified by affinity chromatography. The binding of porcine hnRNPK and different mutants of c-Src protein were verified via GST pull-down assay in vitro. The results showed that the yeast two-hybrid expressing vectors of porcine hnRNPK and different mutants of c-Src were successfully constructed. The bait and prey plasmids were transformed into yeast cells and proved to have no self-activation activity in yeast cells. In yeast, the yeast colonies co-transformed with pGBKT7-hnRNPK and pGADT7-c-Src or pGADT7-ΔPTKc or pGADT7-SH3 grew well in SD-Trp -Leu -His-Ade medium compared with negative controls, indicating that porcine hnRNPK directly interacted with SH3 domain at c-Src protein N-terminal in yeast. The expression vectors of porcine hnRNPK and different mutants of c-Src were successfully constructed, and the soluble His-hnRNPK, GST-c-Src, GST-ΔSH3, GST-ΔSH2, GST-ΔPTKc and GST-SH3 fusion proteins were obtained via induction expression by IPTG and purification. The further GST pull-down assay showed strong interaction between porcine hnRNPK protein and N-terminal of c-Src protein, but weak interaction between hnRNPK protein and SH2, PTKc domains of c-Src protein. The results indicate that porcine hnRNPK protein can interact with the N-terminal domain (SH3 domain) of c-Src protein, which is convenient for studying the regulation sites of porcine c-Src protein, and find a technical platform for researching the mechanism of hnRNPK protein regulated kinase activity of c-Src protein.

CLC Number:

S828.2

LIANG Xiao-juan, XU Hai-xia, LI Rui, ZHANG Peng-peng, XU Yong-jie. Interaction Analysis between Porcine hnRNPK and Tyrosine Protein Kinase c-Src[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 243-252.

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Interaction Analysis between Porcine hnRNPK and Tyrosine Protein Kinase c-Src

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