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

Highlights

• PLAC1 is a new-identified cancer testis antigen.

• PLAC1 presents prevalent expression in cancer tissues and cells.

• Recombinant full length PLAC1 was over-expressed and purified in E. coli.

• Full length PLAC1 was produced by using cold shock protein and Rosetta cells.

• A mild denaturant reagent was used to preserve the 2 and 3 structure of 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.

Introduction

Placenta-specific 1 (PLAC1) is a small (212 amino acid residues) membrane-associated protein [1] involved in placental development and its normal expression is almost restricted to placental trophoblast cells [2]. PLAC1-between different mammalian species - consists of (1)a conserved signal peptide from residues 1 to 23, (2) a number of well-conserved residues in transmembrane domain (TMD) (residues 20–50) and (3) an extremely conserved area in the extracellular space (residues 58–118) that is homologous to the N-terminal sub-domain of the zona pellucida (ZP3) glycoprotein [3], [4]. PLAC1 is normally expressed in the apical villous surface of syncytiotrophoblasts and proposed to be involved in placenta anchoring to the endometrium that maintaining the contact during gestation [2]. Furthermore, the strong protein binding interaction was proposed to occur in ZP3-like extracellular domain [5].

The strong expression of PLAC1 was demonstrated in several human solid tumors including; non small cell lung [6], breast [4], hepatocellular, colorectal [7], [8], and gastric cancers [9], suggesting it has a role as an oncoplacental protein [10]. Recently, we observed that PLAC1 is differentially expressed in prostate cancer and has a strong positive association with Gleason score making it a potential protein target for immunotherapy of prostate cancer [11]. This molecule also was proposed as an attractive target for a set of cancers from histologically different origins [12].

Membrane proteins (MPs) are classified as the major set of protein drug targets due to their essential biological functions in the body [13], [14], [15]. Around 25% of all genes in both prokaryotes and eukaryotes including PLAC1 encode for MPs [16]. Structural study and characterization of MPs call for an efficient expression systems yielding reasonable amount of protein in its native form. Cellular expression of recombinant membrane proteins, however, regularly outcomes in protein aggregation and misfolding that can be due to the hydrophobic properties of transmembrane parts. Expression of proteins at low levels or in insoluble form is considered a major challenging issue in structural biology [17]. In most instances, it is due to the inappropriate folding of human proteins in Escherichia coli cells, where they can be digested by proteases or accumulated as inclusion bodies (IBs) [17]. The poor protein expression has been demonstrated to be improved by gene manipulation [18], [19]. On the other hand, the production of correctly-folded protein is reported to be more complicated and needs a balance between the rate of gene expression and the solubility of protein [20]. To improve proper protein folding and increase the yield of soluble protein, a variety of strategies have been established; 1) overproduction of recombinant proteins at low temperature; it has been reported that the temperature affects the rate of protein synthesis, folding kinetics [21], [22] and the hydrophobic interactions involved in self aggregation as well as protein degradation [23]. Nonetheless, protein expression at low temperature can present some disadvantages, including reduced replication, transcription, and translation rates. These limitations have been proven to be avoided by using cold-inducible promoters such as cold shock promoter cspA [16], [17], [18] that optimize the rate of protein production at low temperature [24]. 2) Optimization of the cultivation conditions; the composition of the cell growth medium and the fermentation variables are important for the prevention of protein aggregation. A careful optimization has been shown to significantly improve the yield and quality of soluble protein production. 3) The expression of the protein of interest in fusion with recombinant tags such as thioredoxin (TRX) and glutathione-S-transferase (GST) [8], [9], [10]. Fusion partners are very stable peptide or protein molecules genetically connected to target proteins for optimization of the solubility and purification. 4) The use of E. coli-engineered host strains; introduction of DNA mutations in E. coli strains has been reported to alter protein synthesis, degradation, secretion, or folding [25]. The expression of proteins in oxidative cytoplasm of Origami or Rosettagami cells [26], [27], where the formation of the disulfide bonds is more favored, improves folding of disulfide-containing proteins. And finally 5) Co-production of molecular chaperones and folding mediators [11]; the initial folding of proteins can be accompanied by molecular chaperones that avoid protein aggregation via binding to exposed hydrophobic areas on misfolded polypeptides, and transferring molecules to their specific sub-cellular target. Although, the expression of PLAC1 gene has been demonstrated in almost one hundred cancer cell lines standing for fourteen distinct cancers [4], [6], [7], its functional behavior remains unclear due to the lack of the structural information. Therefore, recombinant production of PLAC1 can be an attractive strategy to obtain large amounts of protein for further bioresearch applications. Here, we have demonstrated the expression of full length and truncated form of PLAC1 in E. coli and tried to optimize solubility of the recombinant protein using four out of five strategies mentioned above.