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Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells

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Abstract

Objectives

Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein.

Results

HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media.

Conclusions

RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.

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Acknowledgments

This study was a part of the dissertation of Hajar Jaberie, submitted to Shiraz University of Medical Sciences in partial fulfillment of the requirements for the Ph. D in clinical biochemistry. This work was supported by a grant from the Vice Chancellor for Research, Shiraz University of Medical Sciences (Grant Number 92-6699).

Supporting information

Supplementary Table 1—Primers’ sequence used for the related RT-PCR reactions.

Supplementary Fig. 1—The 1257 base pair full length A1AT RT-PCR product (panel A, lane 1) was cloned into pcDNA3.1 expression vector. The presence of the insert was confirmed by digestion of pcDNA3.1-A1AT construct with HindIII/EcoRI restriction enzymes (panel B, lane 2). M: DNA size marker

Author information

Authors and Affiliations

  1. Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Zand Street, Shiraz, Iran

    Hajar Jaberie & Fakhraddin Naghibalhossaini

  2. Autoimmune Research Center and Recombinant Protein Laboratory, Shiraz University of Medical Sciences, Shiraz, Iran

    Fakhraddin Naghibalhossaini

Authors
  1. Hajar Jaberie
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  2. Fakhraddin Naghibalhossaini
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Correspondence to Fakhraddin Naghibalhossaini.

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Supplementary material 1 (DOCX 12 kb)

10529_2016_2150_MOESM2_ESM.tif

Supplementary material 2 (TIFF 202 kb) Supplementary Fig. 1. The1257 base pair full length A1AT RT-PCR product (panel A, lane 1) cloned into pcDNA3.1 expression vector. The presence of the insert was confirmed by digestion of pcDNA3.1-A1AT construct with HindIII/EcoRI restriction enzymes (panel B, lane 2). M: DNA size marker

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Jaberie, H., Naghibalhossaini, F. Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells. Biotechnol Lett 38, 1683–1690 (2016). https://doi.org/10.1007/s10529-016-2150-z

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  • DOI: https://doi.org/10.1007/s10529-016-2150-z

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