Expression and Characterization of a Single-Chain Variable Fragment against Human LOX-1 in Escherichia coli and Brevibacillus choshinensis
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The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and response surface methodology, under which the yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature, and particle diameter size were 1.01E-07 M, 55.2 ± 0.3ºC, and 9.388 nm for the scFv expressed by B. choshinensis, and 4.53E-07 M, 52.5 ± 0.3ºC, and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.
Abstract
The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and response surface methodology, under which the yield reached up to 1.5 g/l in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature, and particle diameter size were 1.01E-07 M, 55.2 ± 0.3ºC, and 9.388 nm for the scFv expressed by B. choshinensis, and 4.53E-07 M, 52.5 ± 0.3ºC, and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.
참고문헌 (32)
- Sawamura T, "An endothelial receptor for oxidized low-density lipoprotein", Nature, 386, pp.73~77 (1997)
- Ding Z, "LOX-1, oxidant stress, mtDNA damage, autophagy, and immune response in atherosclerosis", Can. J. Physiol. Pharmacol, 92, pp.524~530 (2014)
- Xu S, "LOX-1in atherosclerosis: biological functions and pharmacological modifiers", Cell. Mol. Life Sci, 70, pp.2859~2872 (2013)
- Murase T, "Identification of soluble forms of lectin-like oxidized LDL receptor-1. Arterioscler", Thromb. Vasc. Biol, 20, pp.715~720 (2000)
- Shaw DJ, "The role of lectin-like oxidised low-density lipoprotein receptor-1 in vascular pathology", Diab. Vasc. Dis. Res, 11, pp.410~418 (2014)
- Li D, "Molecular imaging of atherosclerotic plaques targeted to oxidized LDL receptor LOX-1 by SPECT", CT and magnetic resonance, 2010, pp.3, 464~472
- Yamada T., "Therapeutic monoclonal antibodies", Keio J. Med, 60, pp.37~46 (2011)
- Andersen DC, "Production technologies for monoclonal antibodies and their fragments", Curr. Opin. Biotechnol, 15, pp.456~462 (2004)
- Tokunaga M, "Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner", Appl. Microbiol. Biotechnol, 97, pp.8569~8580 (2013)
- Ahmad ZA, "scFv antibody: principles and clinical application", Clin. Dev. Immunol, 2012, pp.980250~ (2012)
- Iwamoto S, "An alternative protein standard to measure activity of LOX-1 ligand containing apoB (LAB) -utilization of anti-LOX-1 single-chain antibody fused to apoB fragment", J. Atheroscler. Thromb, 18, pp.818~828 (2011)
- Babaei, Arash, "Production of a Recombinant Anti-Human CD4 Single-Chain Variable- Fragment Antibody Using Phage Display Technology and Its Expression in Escherichia coli", Journal of Microbiology and Biotechnology, 21(5), pp.529~535 (2011), 한국미생물·생명공학회
- F. Shahryari, "Generation and Expression in Plants of a Single-Chain Variable Fragment Antibody Against the Immunodominant Membrane Protein of Candidatus Phytoplasma Aurantifolia", Journal of Microbiology and Biotechnology, 23(8), pp.1047~1054 (2013), 한국미생물·생명공학회[DOI]
- Groff D, "Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins", MAbs, 6, pp.671~678 (2014)
- Liu A, "Expression of V(H)-linker-V(L) orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B1 in Escherichia coli", J. Ind. Microbiol. Biotechnol, 42, pp.255~262 (2015)
- Chen C, "High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain", Biotechnol. Bioeng, 85, pp.463~474 (2004)
- Mu T, "Efficient soluble expression of secreted matrix metalloproteinase 26 in Brevibacillus choshinensis", Protein Expr. Purif, 91, pp.125~133 (2013)
- Choy CJ, "A method to determine the mode of binding for GCPII inhibitors using bio-layer interferometry", J. Enzyme Inhib. Med. Chem, 31, pp.1690~1693 (2016)
- Yang Liu, "Effects of Temperature and Additives on the Thermal Stability of Glucoamylase from Aspergillus niger", Journal of Microbiology and Biotechnology, 25(1), pp.33~43 (2015), 한국미생물·생명공학회[DOI]
- Gao YS, "Sequential events in the irreversible thermal denaturation of human brain-type creatine kinase by spectroscopic methods", Int. J. Mol. Sci, 11, pp.2584~2596 (2010)
- Cheng YM, "Functional expression of recombinant human trefoil factor 1 by Escherichia coli and Brevibacillus choshinensis", BMC Biotechnol, 15, pp.32~ (2015)
- Onishi H, "Efficient production of anti-fluorescein and anti-lysozyme as single-chain anti-body fragments (scFv) by Brevibacillus expression system", Protein Expr. Purif, 91, pp.184~191 (2013)
- Hanagata H, "Efficient expression of antibody fragments with the Brevibacillus expression system", Antibodies, 3, pp.242~252 (2014)
- D’Urzo N, "High-level intracellular expression of heterologous proteins in Brevibacillus choshinensis SP3 under the control of a xylose inducible promoter", Microb. Cell Fact, 12, pp.12~ (2013)
- Maehashi K, "Extracellular production of riboflavin-binding protein, a potential bitter inhibitor, by Brevibacillus choshinensis", Protein Expr. Purif, 71, pp.85~90 (2010)
- Zou C, "Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis", J. Ind. Microbiol. Biotechnol, 43, pp.495~504 (2016)
- Gaciarz A, "Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli", Microb. Cell Fact, 15, pp.22~ (2016)
- Tsybovsky Y, "Folding of an antibody variable domain in two functional conformations in vitro: calorimetric and spectroscopic study of the anti-ferritin antibody VL domain", Protein Eng. Des. Sel, 20, pp.481~490 (2007)
- Baden EM, "Altered dimer interface decreases stability in an amyloidogenic protein", J. Biol. Chem, 283, pp.15853~15860 (2008)
- Panchal J, "Analyzing subvisible particles in protein drug products: a comparison of dynamic light scattering (DLS) and resonant mass measurement (RMM)", AAPS J, 16, pp.440~451 (2014)
- Jonasson P, "Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli", Biotechnol. Appl. Biochem, 35, pp.91~105 (2002)
- Turoverov KK, "Ultra-violet fluorescence of actin. Determination of native actin content in actin preparations", FEBS Lett, 62, pp.4~6 (1976)