Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1)

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.

Placenta-specific1 (PLAC1) is a potential target for antibody-drug conjugate-based prostate cancer immunotherapy

“…The recombinant full length extracellular domain of human PLAC1 (rhPLAC1) was produced as described elsewhere 42 . More information is provided in supplementary information file.…”

Optimization of Expression and Purification of Recombinant Mouse plac1

“…Expression of mouse plac1 was performed as described earlier 12 . In brief, after confirming the sequence of the final minipreps by sequencing, they were transformed into the four E. coli strains (Novagen, USA), namely BL21 (DE3), Origami (DE3), Rosettagami (DE3) pLysS, and Shuffle T7 (DE3).…”

“…Human PLAC1 expression was already optimized in prokaryotic systems producing PLAC1 with high purity and yield 12 . Although human and mouse PLAC1 share common epitopes and may induce cross-reactive humoral immune responses, in vitro studies in murine cancer models need host-specific proteins.…”

Development of a ZHER3‐Affibody‐Targeted Nano‐Vector for Gene Delivery to HER3‐Overexpressed Breast Cancer Cells

“…The proteins were purified by Ni Sepharose High Performance (GE healthcare, UK) under denaturing conditions. As previously reported, all situations of protein purification were checked. The cells were collected, lysed in (Tris (100 m m ), phenylmethylsulfonyl fluoride (PMSF) (1 m m ), urea (2 m ), and NaCl (500 m m ), pH 12.0), and centrifuged for 40 min to pellet the insoluble fraction.…”

“…The purity of peptides was analyzed by western blot using a horseradish peroxidase (HRP)‐conjugated monoclonal anti‐His antibody (Roche) at a 1:100000 dilution and with sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) stained with Coomassie brilliant blue R‐250 (Bio‐Rad). The protein concentration was determined by the Bradford protein assay method using BSA as the standard or by spectroscopy using a calculated molar absorption coefficient at 280 nm (e280) of 18 450, 6990, and 12 950 m −1 cm −1 for the purified protein RALA‐HER3, RALA‐Taq, and ZHER3, respectively.…”