Title
Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1)
Author
Mahboobeh Nazari, Amir-hassan Zarnani, Roya Ghods, Rahman Emamzadeh, Somayeh Najafzadeh, Arash Minai-Tehrani, Jafar Mahmoudian, Maryam Yousefi, Sedigheh Vafaei, Sam Massahi, Mohammad-Reza Nejadmoghaddam
Year
2017
Journal
Protein Expression and Purification
Abstract
Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Biochemistry