Skip to main content
SpringerLink
Log in

Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

With microplate-immobilized polyclonal antibodies against a starting enzyme or its active mutant bearing consistent accessible epitopes, the maximum activity of an adsorbed enzyme/mutant (Vs) was predicted for comparison to recognize weakly-positive mutants. Rabbit antisera against Escherichia coli alkaline phosphatase (ECAP) were fractionated with 33% ammonium sulfate to yield crude polyclonal antibodies for conventional immobilization in 96-well microplates. The response curve of the activities of ECAP/mutant adsorbed by the immobilized polyclonal antibodies to protein quantities from a cell lysate was fit to an approximation model to predict Vs. With 0.4 μg crude polyclonal antibody for immobilization, Vs was consistent for ECAP in cell lysates bearing fourfold differences in its apparent specific activities when its abundance was greater than 0.9%. The ratio of Vs of the mutant R168K to that of ECAP was 1.5 ± 0.1 (n = 2), consistent with that of their specific activities after affinity purification. Unfortunately, the prediction of Vs with polyclonal antibodies that saturated microplate wells was ineffective to Pseudomonas aeruginosa arylsulfatase bearing less than 2% specific activity of ECAP. Therefore, with microplate-immobilized polyclonal antibodies to adsorb enzyme/mutants from cell lysates, high-throughput prediction of Vs was practical to recognize weakly-positive mutants of starting enzymes bearing fairly-high activities.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

BSA:

Bovine serum albumin

ECAP:

Escherichia coli alkaline phosphatase

ELISA:

Enzyme-linked-immunoabsorbent-assay

HTP:

High-throughput

PAAS:

Pseudomonas Aeruginosa arylsulfatase

Tris:

Tris-(hydroxymethyl)-aminomethane

Vs:

The maximum adsorption activity

References

  1. Kiss G, Celebi-Olcum N, Moretti R, Baker D, Houk KN (2013) Computational enzyme design. Angew Chem Int Ed Engl 52:5700–5725

    Article  CAS  Google Scholar 

  2. Zanghellini A (2014) De novo computational enzyme design. Curr Opin Biotechnol 29:132–138

    Article  CAS  Google Scholar 

  3. Bommarius AS (2015) Biocatalysis: a status report. Annu Rev Chem Biomol Eng 6:319–345

    Article  CAS  Google Scholar 

  4. Packer MS, Liu DR (2015) Methods for the directed evolution of proteins. Nat Rev Genet 16:379–394

    Article  CAS  Google Scholar 

  5. Reetz MT (2016) Directed evolution of selective enzymes: catalysts for organic chemistry and biotechnology. Wiley, Weinheim

    Book  Google Scholar 

  6. Feng J, Liu H, Yang X, Gao A, Liao J, Feng L, Pu J, Xie Y, Long G, Li Y, Liao F (2013) Comparison of activity indexes for recognizing enzyme mutants of higher activity with uricase as model. Chem Cent J 7:69

    Article  Google Scholar 

  7. Yang W, Xue L, Fang L, Chen X, Zhan C-G (2010) Characterization of a high-activity mutant of human butyrylcholinesterase against (−)-cocaine. Chem Biol Interact 187:148–152

    Article  CAS  Google Scholar 

  8. Li Y, Yang X, He C, Hu X, Pu J, Liu L, Long G, Liao F (2014) Facile quantitative comparison of specific activities of fusion-tagged enzyme/mutants in cell lysates via prediction of their maximum adsorption by anti-tag antibody immobilized in microplate wells. RSC Adv 4:29925–29932

    Article  CAS  Google Scholar 

  9. Li Y, Long G, Yang X, Hu X, Feng Y, Tan D, Xie Y, Pu J, Liao F (2015) Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities. Int J Biol Macromol 74:211–217

    Article  CAS  Google Scholar 

  10. Feng J, Wang L, Liu H, Yang X, Liu L, Xie Y, Liu M, Zhao Y, Li X, Wang D, Zhan C-G, Liao F (2015) Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions. Appl Microbiol Biotechnol 99:7973–7986

    Article  CAS  Google Scholar 

  11. Blong RM, Bedows E, Lockridge O (1997) Tetramerization domain of human butyrylcholinesterase is at the C-terminus. Biochem J 327:747–757

    Article  CAS  Google Scholar 

  12. Reetz MT (2013) The importance of additive and non-additive mutational effects in protein engineering. Angew Chem Int Ed Engl 52:2658–2666

    Article  CAS  Google Scholar 

  13. Acevedo-Rocha CG, Hoebenreich S, Reetz MT (2014) Iterative saturation mutagenesis: a powerful approach to engineer proteins by systematically simulating Darwinian evolution. Methods Mol Biol 1179:103–128

    Article  Google Scholar 

  14. Privett HK, Kiss G, Lee TM, Blomberg R, Chica RA, Thomas LM, Hilvert D, Houk KN, Mayo SL (2012) Iterative approach to computational enzyme design. Proc Natl Acad Sci USA 109:3790–3795

    Article  CAS  Google Scholar 

  15. Chen Z, Ma Y, He M, Ren H, Zhou S, Lai D, Wang Z, Jiang L (2015) Semi-rational directed evolution of monoamine oxidase for kinetic resolution of rac-mexiletine. Appl Biochem Biotechnol 176:2267–2278

    Article  CAS  Google Scholar 

  16. Liu H, Yang X, Liu L, Dang J, Xie Y, Zhang Y, Pu J, Long G, Li Y, Yuan Y, Liao J, Liao F (2013) Spectrophotometric-dual-enzyme-simultaneous-assay in one reaction solution (SDESA): chemometrics and experimental models. Anal Chem 85:2143–2154

    Article  CAS  Google Scholar 

  17. Liu H, Yuan M, Yang X, Hu X, Dang J, Xie Y, Pu J, Li Y, Zhan C-G, Liao F (2015) Comparison of candidate pairs of hydrolytic enzymes for spectrophotometric-dual-enzyme-simultaneous-assay. Anal Sci 31:421–427

    Article  CAS  Google Scholar 

  18. Yuan M, Yang X, Li Y, Liu H, Pu J, Zhan C-G, Liao F (2016) Facile alkaline lysis of Escherichia coli cells in high-throughput mode for screening enzyme mutants: arylsulfatase as an Example. Appl Biochem Biotechnol 179:545–557

    Article  CAS  Google Scholar 

  19. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  20. Pu J, Hu X, Liao J, Li Y, Qin J, Xie Y, Zhan C-G, Yang X, Liao F (2016) Achievement of linear response for competitive bioaffinity assay of ligand: criteria of optimized interaction system. RSC Adv 6:110856–111865

    Google Scholar 

  21. Feng T, Yang X, Wang D, Hu X, Liao J, Pu J, Zhao X, Zhan C-G, Liao F (2017) A practical system for high-throughput screening of mutants of bacillus fastidiosus uricase. Appl Biochem Biotechnol 181:667–681

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (Nos. 31570862, and 21402108), and the Education Ministry of China (No. 20125503110007).

Author information

Author notes

    Authors and Affiliations

    1. Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China

      Yiran Feng, Xiaolan Yang, Deqiang Wang, Xiaolei Hu, Huimin Chong & Fei Liao

    2. Central Laboratory, Yongchuan Hospital, Chongqing Medical University, No. 439, Xuanhua Road, Yongchuan, Chongqing, 402160, China

      Juan Liao

    3. Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY, 40536, USA

      Chang-guo Zhan

    Authors
    1. Yiran Feng
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Xiaolan Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Deqiang Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Xiaolei Hu
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Huimin Chong
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Juan Liao
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Chang-guo Zhan
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Fei Liao
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Fei Liao.

    Ethics declarations

    Conflict of interest

    The authors declare that they have no conflicts of interest.

    Ethical Approval

    This article does not contain any studies with human participants or animals performed by any of the authors.

    Additional information

    Yiran Feng and Xiaolan Yang have contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Feng, Y., Yang, X., Wang, D. et al. Polyclonal Antibodies in Microplates to Predict the Maximum Adsorption Activities of Enzyme/Mutants from Cell Lysates. Protein J 36, 212–219 (2017). https://doi.org/10.1007/s10930-017-9716-z

    Download citation

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10930-017-9716-z

    Keywords

    Search

    Navigation