Codon optimization and expression of irisin in Pichia pastoris GS115.
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.
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Huikun
H
Duan
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Haisong
H
Wang
Tianjin Children's Hospital, 300074 Tianjin, China.
Baicheng
B
Ma
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Pingzhe
P
Jiang
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Peipei
P
Tu
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Zaizhong
Z
Ni
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Xiaodan
X
Li
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Miao
M
Li
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Xiaofeng
X
Ma
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China.
Bin
B
Wang
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China.
Ri
R
Wu
State Key Laboratory of Medicinal Chemical Biology, Nankai University, 300071 Tianjin, China; Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life Science, Nankai University, 300071 Tianjin, China. Electronic address: mgl@nankai.edu.cn.
Minggang
M
Li
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D018274:Electroporation; D005353:Fibronectins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000072235:Mating Factor; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013696:Temperature
D003062:Codon; C577593:FNDC5 protein, human; D005353:Fibronectins; D010455:Peptides; D011994:Recombinant Proteins; D000072235:Mating Factor; D000432:Methanol
Codon optimized; Expression; Irisin