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High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics

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Abstract

Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2–20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in E. coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify > 95% pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and recombinant light chain A suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.

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Abbreviations

BoNT:

Botulinum neurotoxin

BoNT/A:

Botulinum neurotoxin Type A

DTT:

Dithiothreitol

DNA:

Deoxyribonucleic acid

DrBoNT/A:

Deactivated botulinum neurotoxin type A

E.coli :

Escherichia coli

EDTA:

Ethylenediaminetetraacetic acid

FRET:

Fluorescence resonance energy transfer

GST:

Glutathione-S-transferase

HC:

Heavy chain

IPTG:

Isopropyl-b-d-thiogalactopyranoside

LB:

Luria–Bertani

LC:

Light chain

PCR:

Polymerase chain reaction

RBD:

Receptor binding domain

rLC-TD/A:

Recombinant light chain-translocation domain

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SNAP-25:

Synaptosome associated protein of 25 kDa

SNARE:

Soluble N-ethylmaleimide-sensitive factor attachment protein

TD:

Translocation domain

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Acknowledgements

This work was supported in part by a grant from the National Institute of Allergy and Infectious Diseases (NIAID—1U01A1078070-02) and by Maryada Foundation. The authors would like to thank Mr. Stephen J. Riding for his assistance in purification procedures. The authors also thank Dr. Gowri Chellappan for her assistance in liposome preparation procedures.

Author information

Author notes

  1. Nagarajan Thiruvanakarasu

    Present address: US Food and Drug Administration, Silver Spring, MD, USA

  2. Harkiranpreet Kaur Dhaliwal

    Present address: Department of Pharmaceutical Sciences, Northeastern University, Boston, 02115, USA

Authors and Affiliations

  1. Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, North Dartmouth, MA, 02747, USA

    Harkiranpreet Kaur Dhaliwal & Ghuncha Ambrin

  2. Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, North Dartmouth, MA, 02747, USA

    Nagarajan Thiruvanakarasu, Kruti Patel & Shouwei Cai

  3. Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA, 02747, USA

    Raj Kumar & Bal Ram Singh

Authors
  1. Harkiranpreet Kaur Dhaliwal
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  3. Raj Kumar
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Contributions

Conceived the idea: NT, HD, BS. Designed and carried out the experiments: HD, NT, RK. Kinetics experiments: KP, GA. Analyzed the data: HD, RK, BS. Contributed reagents/materials/analysis tools: SC, BS. Offered comments on data interpretation and manuscript preparation: SC, RK, BS. Wrote the manuscript: HD, BS.

Corresponding author

Correspondence to Bal Ram Singh.

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Dhaliwal, H.K., Thiruvanakarasu, N., Kumar, R. et al. High Yield Preparation of Functionally Active Catalytic-Translocation Domain Module of Botulinum Neurotoxin Type A That Exhibits Uniquely Different Enzyme Kinetics. Protein J 36, 489–501 (2017). https://doi.org/10.1007/s10930-017-9744-8

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