Biochemical and Biophysical Research Communications
Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility
Introduction
The epidermal growth factor receptor (EGFR) is a tyrosine kinase family (ErbBs) cell-surface receptor involved in cell proliferation, differentiation, and survival [1]. Mutations in the EGFR gene have been associated with several types of tumors [1], and therapeutic Anti-EGFR monoclonal antibodies (mAbs) such as Panitumumab and Cetuximab have been developed [2,3]. Current mAbs have limitations such as their low tumor penetration, slow blood clearance rate and especially, and their high production cost [4,5]. Recombinant antibody fragments such as ScFv [4] and VHH [6] are much smaller than the full-length mAbs and are expected to address these issues, and additionally to have a rapid tumor penetration rate [7]. ScFv consists of the variable heavy (VH) and light (VL) domains linked by an artificial flexible polypeptide, and its molecular weight is merely ∼28 kDa, which is considerably smaller than the full-length antibodies (∼150 kDa).
Although ScFv has been produced in various expression systems including mammalian [8], yeast [9] and bacterial cells [10], ScFv remains in many instances limited by solubility issues. In particular, ScFv forms inclusion bodies when it is overexpressed in E. coli [10], which is the most popular and common expression system. The solubility of ScFv has been enhanced by fusion to highly soluble proteins such as thioredoxin [11], N-utilization substance (NusA), maltose binding protein (MBP) [12] and small ubiquitin-like modifier (SUMO) [13]. However, due to their large sizes, the fusion proteins often interfere with ScFv's binding to the antigens and need to be removed during purification, reducing its solubility [12].
In this study, we applied our Solubility Enhancing Peptide (SEP) tag strategy [14,15] to increase the solubility of Anti-EGFR ScFv. To this end, we attached a short 12-residue SEP tag (C9R) to the C termini of Anti-EGFR ScFv. The SEP tag increased the solubility of Anti-EGFR ScFv, both during and after purification, without affecting the EGFR binding activity. In addition, the SEP tag unexpectedly increased the yield of Anti-EGFR ScFv by threefold, and we purified 17.15 mg of active Anti-EGFR ScFv-C9R from a 200 mL LB culture.
Section snippets
Computational model
Anti-EGFR ScFv with a SEP tag was constructed with Modeller [16] using ScFv PL-2 as a template (PDB ID 5KOV-chain C). In order to take into account the protein's internal motions in the Brownian dynamics (BD) calculation, where the proteins are treated as a rigid-body particle, we generated 10 Anti-EGFR ScFv structures by all-atom molecular dynamics (MD) simulation. The MD simulation was carried out for 30 ns using Gromacs 4.7 [17] on an HPC5000 server equipped with a NvidiaTesla K40 GPU board
Prediction of the mutant's solubility
Prior to experimental assessments, we predicted the effects of attaching a SEP tag to the C terminus of Anti-EGFR ScFv on its solubility, because we suspected that the solubilization of ScFv, which is a two-domain protein with two disulfide bonds, might be more complex than solubilizing single-domain proteins [14,15]. The solubility of Anti-EGFR ScFv variants was predicted using SDA 7.1, which allows simulating the trajectories of several tens of rigid body proteins and estimate the formation
Funding
This study was supported by a research grant from the TaNeDS global research program and JSPS grant-in-aid for scientific research grants (KAKENHI-21300110, 15H04359, and 18H02385) to Y.K.
Conflicts of interest
None.
Acknowledgments
We thank the members of the Kuroda Laboratory for discussion and suggestions, Dr. Punitha Velmurugan (JSPS) and Dr. Richa Tambi (JSPS) for their kind help with manuscript preparation. We are grateful to Mr. Katsuhiko Hirose and Mr. Satoshi Kosuda for support with MD and BD calculations, Mr. Hiromitsu Sakurai for preliminary experiments, and Ms. Patricia McGahan for English proofreading.
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