Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Cenk DAĞLIOĞLU

Article Details

Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Authors:

Cenk DAĞLIOĞLU

Publisher:

TUBITAK

Main Topics:

Life sciences

Journal:

Turkish Journal of Biology 2017 Vol.41 Issue 5, pp.746-753

Document Type:

Article

Publication Date:

9/2017

Document Language:

English

Document ID (Org No.):

2000811331

Frequency:

Bimonthly

eISSN:

1303-6092

pISSN:

1300-0152

Abstract:

 

The yeast Pichia pastorisexpression system was investigated for the production of human cathepsin C (CatC) recombinant protein. The full-length CatC cDNA, corresponding to amino acids 12-475, was synthesized from interleukin-2 (IL-2) stimulated human peripheral blood mononuclear cells and subcloned in the pGEM-T cloning vector. After confirming the DNA sequence of the insert, the gene was cloned into the pPICZ\alphaA expression vector under the control of the methanol-inducible alcohol oxidase (AOX1) promoter and transformed to P. pastoris X-33 cells. The expressed protein was secreted into the culture medium through the alpha-factor mating signal sequence of the expression vector. Analysis of the culture supernatant revealed that the recombinant human CatC was secreted as a 58-kDa molecule, indicating that human CatC was accumulated in the culture supernatant as proform composed of the residual propart, the activation peptide, and the heavy and light chains. Extracellular recombinant proCatC was further activated by cysteine endoprotease papain in vitro and its activity was confirmed by assays using a synthetic substrate.

Article Details

Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Authors: Cenk DAĞLIOĞLU

Publisher: TUBITAK

Main Topics: Life sciences

Journal: Turkish Journal of Biology 2017 Vol.41 Issue 5, pp.746-753

Document Type: Article

Publication Date: 9/2017

Document Language: English

Document ID (Org No.): 2000811331

Frequency: Bimonthly

eISSN: 1303-6092

pISSN: 1300-0152

Issues

Authors:

Cenk DAĞLIOĞLU

Publisher:

TUBITAK

Main Topics:

Life sciences

Journal:

Turkish Journal of Biology 2017 Vol.41 Issue 5, pp.746-753

Document Type:

Article

Publication Date:

9/2017

Document Language:

English

Document ID (Org No.):

2000811331

Frequency:

Bimonthly

eISSN:

1303-6092

pISSN:

1300-0152