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Oxidative Folding of Conopeptides Modified by Conus Protein Disulfide Isomerase

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Abstract

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis–trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis–trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.

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Abbreviations

PDI:

Protein disulfide isomerase

sPDI:

Conus protein disulfide isomerase

HPLC:

High performance liquid chromatography

MS:

Mass spectrometry

PPIase:

Peptidyl-prolyl cis–trans isomerase

LZM:

Lysozyme

CD:

Circular dichroism

SDS-PAGE:

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

TRX:

Thioredoxin

GST:

Glutathione-S-transferase

IPTG:

Isopropyl β -D-1-thiogalactopyranoside

MW:

Molecular weight

PVDF:

Polyvinylidene fluoride

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Acknowledgements

This work was supported by the Natural Science Foundation of Guangdong Province (Grant Number: 2015A030313191); Shenzhen Science and Technology Innovation Committee (Grant Number: ZD201111080120A).

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Author notes

  1. Lei Wang and Xiaomin Wang contributed equally to this study.

Authors and Affiliations

  1. Department of Pharmaceutical Engineering, College of Materials and Energy, South China Agricultural University, Guangzhou, 510642, People’s Republic of China

    Lei Wang

  2. State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, National Engineering Research Center of South China Sea Marine Biotechnology, Department of Biochemistry, College of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, People’s Republic of China

    Lei Wang, Xiaomin Wang, Zhenghua Ren, Wei Tang, Qiong Zou, Jinxing Wang, Han Zhang & Anlong Xu

  3. Research Institute of Sun Yat-sen University in Shenzhen, Shenzhen, 518057, People’s Republic of China

    Shangwu Chen

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  1. Lei Wang
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Correspondence to Lei Wang.

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10930_2017_9738_MOESM1_ESM.docx

Fig.S. The scheme presentation of the expression vectors of pET-28a-sPDI (a) and pETcsPDI-PPI/ pETcsPDI-lt14a (b) (DOCX 60 KB)

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Wang, L., Wang, X., Ren, Z. et al. Oxidative Folding of Conopeptides Modified by Conus Protein Disulfide Isomerase. Protein J 36, 407–416 (2017). https://doi.org/10.1007/s10930-017-9738-6

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