Expression of recombinant human IL-4 in Pichia pastoris and relationship between its glycosylation and biological activity.
Secretory human interleukin 4 (hIL4) is an N-glycosylated pleiotropic cytokine. It is unknown if these N-linked glycans are required and essential for hIL4 protein stability, expression, secretion, and activity in vivo, and hIL4 expressed from Pichia pastoris yeast has not been tested to date. In this study, we successfully expressed human hIL4 in P. pastoris, the methylotrophic yeast, with a yield of 15.0mg/L. Using the site-directed mutagenesis technique, we made two mutant hIL4 cDNA clones (N38A and N105L) and subsequently expressed them in P. pastoris to analyze the relevant function of each N-glycosylation site on hIL4. Our results demonstrate that the glycosylation only occurs at position Asn38, but not Asn105. The glycosylated form of hIL4 unexpectedly has lower biological activity and lower stability when compared to its non-glycosylated form. The implications of this are discussed.
Center for Stem Cell & Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250012, PR China; Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, PR China.
Rui
R
Li
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
Chao
C
Xie
Center for Stem Cell & Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250012, PR China; Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, PR China.
Yuan
Y
Zhang
Center for Stem Cell & Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250012, PR China; Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, PR China.
Bin
B
Li
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
William
W
Donelan
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Shiwu
S
Li
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Shuhong
S
Han
Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, PR China.
Xingli
X
Wang
Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, PR China; Department of Cell Biology and Anatomy, University of South Carolina of Medicine, Columbia, SC 29209, USA. Electronic address: cuitaixing@sdu.edu.cn.
Taixing
T
Cui
Center for Stem Cell & Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250012, PR China; Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, PR China. Electronic address: tangdq@sdu.edu.cn.
Dongqi
D
Tang
D003001:Cloning, Molecular; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015847:Interleukin-4 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D017455:Receptors, IgE / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics*
D006023:Glycoproteins; C508598:IL4 protein, human; D017455:Receptors, IgE; D011994:Recombinant Proteins; D015847:Interleukin-4
N-glycosylation; Pichia pastoris; Recombinant human IL4; Site-directed mutagenesis