One-step purification of a fusion protein of glucagon-like peptide-1 and human serum albumin expressed in pichia pastoris by an immunomagnetic separation technique.
Glucagon-like peptide-1 (GLP-1) has great therapeutic potential to treat diabetes type 2, mainly due to its unique glucose-dependent stimulation of insulin secretion profiles, but its clinical application is limited by its short half-life in vivo, which resultes from degradation by dipeptidyl peptidase IV and/or renal clearance. Developing long-acting GLP-1 analogs is therefore an important step toward using them therapeutically. In this study, the GLP-1/human serum albumin (HSA) fusion protein gene was cloned into the secretor type expression vector pPIC9K and subsequently expressed in Pichia pastoris. The expression quantity reached 58.5 mg/l in small-scale incubation. After optimization and characterization, the GLP-1/HSA fusion protein was successfully purified from the supernatant of the broth using immunomagnetic cellulose microspheres. HPLC showed that the purified GLP-1/HSA had an overall purity of 93.9%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the fusion protein exhibited the expected molecular mass of 70 kDa. Furthermore, that analysis of in vivo activity indicated that GLP-1/HSA reduced the blood glucose level after intraperitoneal administration to Chinese Kunming mice in a dose-dependent manner, and the effects held significantly 4 h after administration. Overall, this study illustrates the development of a long-acting GLP-1/HSA fusion protein expressed in Pichia pastoris.
Chen
Jiaqi
J
Department of Microbiology, College of Life Sciences, Nankai University, Nankai University, Tianjin 30071, China.
Bai
Gang
G
Cao
Yu
Y
Gao
Zhihui
Z
Zhang
Qi
Q
Zhu
Yuanyuan
Y
Yang
Wenbo
W
D000818:Animals; D001786:Blood Glucose / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D052216:Glucagon-Like Peptide 1 / Q000008:administration & dosage / Q000096:biosynthesis / Q000302:isolation & purification*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D051379:Mice; D008815:Mice, Inbred Strains; D010843:Pichia / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000302:isolation & purification*; D012709:Serum Albumin / Q000008:administration & dosage / Q000096:biosynthesis / Q000302:isolation & purification*
D001786:Blood Glucose; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D052216:Glucagon-Like Peptide 1