Development, characterization, and evaluation of a fusion protein of a novel glucagon-like peptide-1 (GLP-1) analog and human serum albumin in Pichia pastoris.
Glucagon-like peptide-1 (GLP-1) has considerable potential as a possible therapeutic agent for type-2 diabetes. Unfortunately, this glucoincretin is short lived due to degradation by dipeptidyl-peptidase IV and rapid clearance by renal filtration. In this study, we attempted to extend GLP-1 action through the attachment of a lysine residue at the N-terminal of GLP-1 (named KGLP-1), and to make a fusion protein with human serum albumin (HSA) in Pichia pastoris. The protein, designated KGLP-1/HSA, was purified by an immunomagnetic separation technique. High performance liquid chromatography (HPLC) showed that the purified protein had an overall purity of 92.0%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the expected molecular mass of 70,297.8 Da. Additionally, the N-terminal sequence of KGLP-1/HSA was confirmed by N-terminal sequencing. The stability and biological activity of KGLP-1/HSA were then evaluated in vitro and in vivo. The findings indicated that fusion KGLP-1/HSA preserved the action of native GLP-1, and the active duration was greatly prolonged.
Gao
Zhihui
Z
Department of Microbiology, College of Life Sciences, Nankai University, Tianjin, China.
Bai
Gang
G
Chen
Jiaqi
J
Zhang
Qi
Q
Pan
Pengwei
P
Bai
Fang
F
Geng
Peng
P
D000818:Animals; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D015870:Gene Expression; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000067757:Glucagon-Like Peptide-1 Receptor; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D051379:Mice; D010843:Pichia / Q000235:genetics*; D055550:Protein Stability; D018027:Receptors, Glucagon / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012709:Serum Albumin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
C000593833:GLP1R protein, human; C000593834:Glp1r protein, mouse; D000067757:Glucagon-Like Peptide-1 Receptor; D007328:Insulin; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D052216:Glucagon-Like Peptide 1; D018819:Dipeptidyl Peptidase 4; D005947:Glucose