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Codon optimization and expression of irisin in Pichia pastoris GS115.
- Source :
-
International journal of biological macromolecules [Int J Biol Macromol] 2015 Aug; Vol. 79, pp. 21-6. Date of Electronic Publication: 2015 Apr 27. - Publication Year :
- 2015
-
Abstract
- Irisin is a novel hormone which is related to many metabolic diseases. In order to illuminate the function and therapeutic effect of irisin, gaining active irisin is necessary. In this work, a codon-optimized irisin gene was designed according to Pichia pastoris synonymous codon usage bias and cloned into the pPIC9K expression vector. Sequencing result indicating that the sequence of irisin was consistent with the modified irisin and the irisin was in frame with α-factor secretion signal ATG. The plasmid pPIC9K-irisin was transformed into GS115 P. pastoris cells through electroporation. The positive transformants were screened on MD medium and analyzed by PCR. Five recombinant GS115/pPIC9K-irisin strains were obtained, but only one strain expressed irisin successfully. SDS-PAGE and Western blot were used to assess the expression level and purity of irisin. The irisin was also simply purified and the effect of pH value, methanol concentration and induction time on the production of irisin was investigated. The results showed that the best conditions of irisin expression were as follows: pH 6.0, 2.0% methanol and induction for 96 h. This work laid the basis for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.<br /> (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Subjects :
- Amino Acid Sequence
Base Sequence
Cloning, Molecular
Electroporation
Fibronectins biosynthesis
Fibronectins isolation & purification
Gene Expression
Humans
Hydrogen-Ion Concentration
Mating Factor
Methanol metabolism
Methanol pharmacology
Molecular Sequence Data
Peptides genetics
Peptides metabolism
Pichia drug effects
Pichia genetics
Pichia metabolism
Plasmids chemistry
Polymerase Chain Reaction
Recombinant Proteins biosynthesis
Recombinant Proteins genetics
Recombinant Proteins isolation & purification
Sequence Analysis, DNA
Temperature
Codon
Fibronectins genetics
Plasmids metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1879-0003
- Volume :
- 79
- Database :
- MEDLINE
- Journal :
- International journal of biological macromolecules
- Publication Type :
- Academic Journal
- Accession number :
- 25931394
- Full Text :
- https://doi.org/10.1016/j.ijbiomac.2015.04.030