EGFR extracellular domain III expressed in Escherichia coli with SEP tag shows improved biophysical and functional properties and generate anti-sera inhibiting cancer cell growth

Brindha, Subbaian; Kibria, Golam; Saotome, Tomonori; Unzai, Satoru; Kuroda, Yutaka

doi:10.1016/j.bbrc.2021.03.102

Cited by 6 publications

References 33 publications

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“…The total expression level of RBD-C9R at 16 °C in LB was 6.25 µg/5ml as estimated from the SDS page gel (Figure 1D), five times higher than for the untagged RBD expressed in the inclusion body. The ability of the C9R tag to increase protein expression is not well understood but is in line with previous observations with EFGR-ECD-III [14], TEV [16]. One-liter expression of RBD-C9R was carried out using the T7 shuffle cell line at 16 °C in LB, and RBD-C9R was purified from the soluble fraction by denaturing Ni-NTA chromatography.…”

Section: Rbd Expression and Purificationsupporting

confidence: 75%

“…Analytical RP-HPLC is a relatively fast and reliable method for analyzing the formation of SS bonds [40,41], and the difference in the retention time can resolve different disulfide bonding patterns in a multi-disulfide bonded protein [16,40,41]. The RP-HPLC elution profiles of the oxidized RBD-C9R expressed in the supernatant of T7 shuffle showed a single peak, suggesting that all of the protein had folded into a single species of S-S bond pairing.…”

Section: Discussionmentioning

confidence: 99%

“…They significantly increase the protein's solubility without much affecting its structure or activity [14,15]. We previously used SEP-tags for improving the solubility and yield of recombinant proteins: EGFR-ECD-III [16], anti-EGFR-ScFv [17], TEV protease [18], and Gaussia luciferase [12].…”

Section: Introductionmentioning

confidence: 99%

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A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

Brindha

Kuroda

2022

IJMS

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An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell’s receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E.coli production of active multi-disulfide-bonded RBD.

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Section: Rbd Expression and Purificationsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

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A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

Brindha

Kuroda

2022

IJMS

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“…To date, the SEP tag is the only way for reliably controlling the protein solubility and aggregation without changing the buffer's condition and without altering the protein's structural and functional properties [38,43,44]. Besides, SEP tags can solubilize recombinant proteins containing multiple disulfide bonds and yield a substantial amount of fully native proteins from E. coli expression systems, which would otherwise not be possible [29,45].…”

Section: The Sep Tag Is a Versatile Technique For Solubilizing Proteinsmentioning

confidence: 99%

“…SEP tag has emerged as a reliable and versatile technique for controlling protein solubility [13,22]. In particular, we have shown their solubilization properties for bovine pancreatic trypsin inhibitor (BPTI) [23], dengue envelope protein [24], anti-epidermal growth factor receptor (EGFR)-ScFv [25], tobacco etch virus (TEV) protease [26], Gaussia luciferase (GLuc) [27,28] and the third domain of EGFR [29]. Here we assessed the effect of arginine and lysine tags to control protein solubility under thermal stress.…”

Section: Introductionmentioning

confidence: 99%

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

et al. 2021

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In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

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EGFR extracellular domain III expressed in Escherichia coli with SEP tag shows improved biophysical and functional properties and generate anti-sera inhibiting cancer cell growth

Cited by 6 publications

References 33 publications

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

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