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Codon optimization and expression of irisin in Pichia pastoris GS115.

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.
AuthorsHuikun Duan, Haisong Wang, Baicheng Ma, Pingzhe Jiang, Peipei Tu, Zaizhong Ni, Xiaodan Li, Miao Li, Xiaofeng Ma, Bin Wang, Ri Wu, Minggang Li
JournalInternational journal of biological macromolecules (Int J Biol Macromol) Vol. 79 Pg. 21-6 (Aug 2015) ISSN: 1879-0003 [Electronic] Netherlands
PMID25931394 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2015 Elsevier B.V. All rights reserved.
Chemical References
  • Codon
  • FNDC5 protein, human
  • Fibronectins
  • Peptides
  • Recombinant Proteins
  • Mating Factor
  • Methanol
Topics
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Codon
  • Electroporation
  • Fibronectins (biosynthesis, genetics, isolation & purification)
  • Gene Expression
  • Humans
  • Hydrogen-Ion Concentration
  • Mating Factor
  • Methanol (metabolism, pharmacology)
  • Molecular Sequence Data
  • Peptides (genetics, metabolism)
  • Pichia (drug effects, genetics, metabolism)
  • Plasmids (chemistry, metabolism)
  • Polymerase Chain Reaction
  • Recombinant Proteins (biosynthesis, genetics, isolation & purification)
  • Sequence Analysis, DNA
  • Temperature

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