Eukaryotic expression, purification, identification, and tissuedistribution of porcine PID1

This study provides a simple and efficient method for yielding a large amount of active recombinant pPID1, which can be useful for further study of the pPID1 protein.

📌 3 Key Takeaways

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Objective & Hypothesis:Phosphotyrosine interaction domain containing 1 (PID1) is a recently discovered gene related to lipid metabolism and may play an important role in fat deposition.

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Key Findings & Data:In this study, in order to scale up the production of the active recombinant porcine PID1 (pPID1) protein, we reported the expression and purification of a His-tagged version of pPID1 in the yeast Pichia pastoris.

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Conclusion & Impact:This study provides a simple and efficient method for yielding a large amount of active recombinant pPID1, which can be useful for further study of the pPID1 protein.

🔬Design:Research Article

📍Scope:Comprehensive Sample

⚙️Variables:Porcine PID1 · Pichia pastoris · purification

Abstract

Phosphotyrosine interaction domain containing 1 (PID1) is a recently discovered gene related to lipid metabolism and may play an important role in fat deposition. In this study, in order to scale up the production of the active recombinant porcine PID1 (pPID1) protein, we reported the expression and purification of a His-tagged version of pPID1 in the yeast Pichia pastoris. The pPID1 cDNA was cloned into the pPICZαA vector and was expressed in methylotrophic yeast (P. pastoris X33) under control of the alcohol oxidase promoter. The intracellularly expressed recombinant protein was purified by Ni-IDA affinity chromatography, yielding over 95% purity and about 1.8 mg/L. The recombinant protein was identified by Western blot. In addition, the tissue distribution of pPID1 protein was investigated, and expression of pPID1 protein was mainly detected in the skeletal muscles and liver. This study provides a simple and efficient method for yielding a large amount of active recombinant pPID1, which can be useful for further study of the pPID1 protein.

Özet

Abstract: Phosphotyrosine interaction domain containing 1 (PID1) is a recently discovered gene related to lipid metabolism and may play an important role in fat deposition. In this study, in order to scale up the production of the active recombinant porcine PID1 (pPID1) protein, we reported the expression and purification of a His-tagged version of pPID1 in the yeast Pichia pastoris. The pPID1 cDNA was cloned into the pPICZαA vector and was expressed in methylotrophic yeast (P. pastoris X33) under control of the alcohol oxidase promoter. The intracellularly expressed recombinant protein was purified by Ni-IDA affinity chromatography, yielding over 95% purity and about 1.8 mg/L. The recombinant protein was identified by Western blot. In addition, the tissue distribution of pPID1 protein was investigated, and expression of pPID1 protein was mainly detected in the skeletal muscles and liver. This study provides a simple and efficient method for yielding a large amount of active recombinant pPID1, which can be useful for further study of the pPID1 protein.

Keywords

Porcine PID1 Pichia pastoris purification tissue distribution

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Cite This Article

CHEN, X., WANG, H., ZHOU, B., HUANG, Z., JIA, G., LIU, G., ZHAO, H. (2015). Eukaryotic expression, purification, identification, and tissuedistribution of porcine PID1. Turkish Journal of Biology, Cilt: 39 - Sayı: 5, 705–711. https://ajindex.com/articles/181476

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References (9)

Canales M, de la Lastra JMP, Naranjo V, Nijhof AM, Hope M, Jongejan F, de la Fuente J (2008). Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris. BMC Biotechnol 8: 14. Caratù G, Allegra D, Bimonte M, Schiattarella GG, D’Ambrosio C, Scaloni A, Napolitano M, Russo T, Zambrano N (2007). Identification of the ligands of protein interaction domains through a functional approach. Mol Cell Proteomics 6: 333– 345. Chen CY, Ai HS, Ren J, Li WB, Li PH, Qiao RM, Ouyang J, Yang M, Ma JW, Huang LS (2011). A global view of porcine transcriptome in three tissues from a full-sib pair with extreme phenotypes in growth and fat deposition by paired-end RNA sequencing. BMC Genomics 12: 448. Chen XL, Huang ZQ, Chen DW, Jia G, Mao XB, Wu XQ (2012a). Role of NYGGF4 in insulin resistance. Mol Biol Rep 39: 5367–5371.
Chen XL, Huang ZQ, Jia G, Guo XL, Tang RY, Wu XQ (2012b). Phosphotyrosine interaction domain containing 1 gene: regulation on meat quality. Chinese J Anim Nutr 24: 591–594.
Chen XL, Huang ZQ, Wang H, Jia G, Liu GM, Zhao H (2013). Porcine phosphotyrosine interaction domain containing 1 modulates 3T3-L1 preadipocyte proliferation and differentiation. Biologia 68: 1010–1014.
Chen XL, Wang H, Huang ZQ, Zhou B, Jia G, Liu GM, Zhao H (2014). Fusion expression of porcine phosphotyrosine interaction domain containing 1 gene and preparation, characterization of its polyclonal antibody. Chinese J Anim Nutr 26: 3349–3355.
Higgins DR, Busser K, Comiskey J, Whittier PS, Purcell TJ, Hoeffler JP (1998). Small vectors for expression based on dominant drug resistance with direct multicopy selection. In: Higgins DR, Cregg JM, editors. Methods in Molecular Biology: Pichia Protocols. Totowa, NJ, USA: Humana Press; pp. 28–41.
Kajiwara Y, Franciosi S, Takahashi N, Krug L, Schmeidler J, Taddel K, Haroutunian V, Fried U, Ehrlich M, Martins RN et al. (2010). Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer’s disease. Mol Neurodegener 5: 1–11.
Kavanaugh WM, Williams LT (1994). An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science 266: 1862–1865.
Li H, Gao X, Kong X, Li S, Xu A, Jin S, Wu D (2011). High level expression of active recombinant human interleukin-3 in Pichia pastoris. Protein Expr Purif 80: 185–193.
Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005