Application of an S-layer protein as a self-aggregating tag for cost-effective separation of recombinant human and yeast d-amino acid oxidases in the aqueous two-phase system

  • Minkyeong Ahn
  • Sangje Park
  • Junhyun Jeon
  • Jung Kyu Choi
  • Yongho KhangEmail author
Original Research Paper



To evaluate whether the surface layer (S-layer) protein of Lactobacillus brevis serves as a self-aggregating protein tag for cost-effective separation of human and yeast d-amino acid oxidases (hDAAO and yDAAO) expressed in E. coli.


In aqueous two-phase (PEG–phosphate) system, the S-layer:DAAO fusion proteins (shDAAO and syDAAO) were separated at the interface with a recovery of 82 ± 10.6% for shDAAO and 95 ± 1.9% for syDAAO. Some shDAAO proteins were separated as precipitates with a recovery of 41 ± 0.5% in phosphate (9%, w/w) using PEG 3000 and PEG 4000 (16%, w/w), while some syDAAO proteins were also isolated as precipitates with a recovery of 75 ± 17.5% in phosphate (9%, w/w) using PEG 4000 and PEG 8000 (16%, w/w).


The S-layer of L. brevis was applied to a self-assembled protein tag to enable cost-effective separation of human and yeast d-amino acid oxidases expressed in E. coli cells. Because of the self-assembling properties of S-layer proteins, human and yeast d-amino acid oxidases fused with S-layer proteins could be easily separated by aggregates at the interface and/or in a few conditions by precipitates to the bottom of the PEG–phosphate aqueous system.


Aqueous two-phase system d-Amino acid oxidase Fusion tag Self-aggregating tag S-layer protein 



This work was supported in part by Yeungnam University.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by MA, SP, JJ, and JKC. The first draft of the manuscript was written by YK and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. Ball J, Gannavaram S, Gadda G (2018) Structural determinants for substrate specificity of flavoenzymes oxidizing d-amino acids. Arch Biochem Biophys 660:87–96. CrossRefPubMedGoogle Scholar
  2. Iqbal M, Tao Y, Xie S, Zhu Y, Chen D, Wang X, Huang L, Peng D, Sattar A, Shabbir MA, Hussain HI, Ahmed S, Yuan Z (2016) Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol Proced Online 18:18. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Jia B, Jeon CO (2016) High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol 6:160196. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Khang YH, Kim IW, Hah YR, Hwangbo JH, Kang KK (2003) Fusion protein of Vitreoscilla hemoglobin with d-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C. Biotechnol Bioeng 82:480–488CrossRefGoogle Scholar
  5. Khang YH, Park HY, Jeong YS, Kim JA, Kim YH (2009) Recombinant S-layer proteins of Lactobacillus brevis mediating antibody adhesion to calf intestine alleviated neonatal diarrhea syndrome. J Microbiol Biotechnol 19:511–519CrossRefGoogle Scholar
  6. Koga R, Miyoshi Y, Sakaue H, Hamase K, Konno R (2017) Mouse d-amino acid oxidase: distribution and physiological substrates. Front Mol Biosci 4:82. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Kondori NR, Paul P, Robbins JP, Liu K, Hildyard JCW, Wells DJ, de Belleroche JS (2018) Focus on the role of d-serine and d-amino acid oxidase in Amyotrophic Lateral Sclerosis/Motor Neuron Disease (ALS). Front Mol Biosci 5:8. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Lin Z, Zhao Q, Zhou B, Xing L, Xu W (2015) Cleavable self-aggregating tags (cSAT) for protein expression and purification. Methods Mol Biol 1258:65–78. CrossRefPubMedGoogle Scholar
  9. Luo G, Yang Q, Yao B, Tian Y, Hou R, Shao A, Li M, Feng Z, Wang W (2019) Slp-coated liposomes for drug delivery and biomedical applications: potential and challenges. Int J Nanomedicine 14:1359–1383. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Molla G (2017) Competitive inhibitors unveil structure/function relationships in human d-amino acid oxidase. Front Mol Biosci 4:80. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Momoi K, Fukui K, Watanabe F, Miyake Y (1988) Molecular cloning and sequence analysis of cDNA encoding human kidney d-amino acid oxidase. FEBS Lett 238:180–184. CrossRefPubMedGoogle Scholar
  12. Pollegioni L, Piubelli L, Sacchi S, Pilone MS, Molla G (2007) Physiological functions of d-amino acid oxidases: from yeast to humans. Cell Mol Life Sci 64:1373–1394. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Pollegioni L, Sacchi S, Murtas G (2018) Human d-amino acid oxidase: structure, function, and regulation. Front Mol Biosci 5:107. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Pum D, Sleytr UB (2014) Reassembly of S-layer proteins. Nanotechnology 25:312001. CrossRefPubMedGoogle Scholar
  15. Seo YM, Khang YH, Yun H (2011) Kinetic resolution of 3-fluoroalanine using a fusion protein of d-amino acid oxidase with Vitroscilla hemoglobin. Biosci Biotechnol Biochem 75:820–822. CrossRefPubMedGoogle Scholar
  16. Shibata C, Iwashita K, Shiraki K (2019) Selective separation method of aggregates from IgG solution by aqueous two-phase system. Protein Expr Purif 161:57–62. CrossRefPubMedGoogle Scholar
  17. Szilágyi B, Ferenczy GG, Keserű GM (2018) Drug discovery strategies and the preclinical development of d-amino-acid oxidase inhibitors as antipsychotic therapies. Expert Opin Drug Discov 13:973–982. CrossRefPubMedGoogle Scholar
  18. Wang W, Sun J, Xiao W, Jiang L, Wang R, Fan J (2017) Change of the N-terminal codon bias combined with tRNA supplementation outperforms the selected fusion tags for production of human d-amino acid oxidase as active inclusion bodies. Biotechnol Lett 39:1733–1740. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyYeungnam UniversityGyeongsanRepublic of Korea

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