ABSTRACT
Glucagon like peptide-1 (GLP-1) regulates glucose mediated-insulin secretion, nutrient accumulation, and ß-cell growth. Despite the potential therapeutic usage for type 2 diabetes (T2D), GLP-1 has a short half-life in vivo (t(1/2) <2 min). In an attempt to prolong half-life, GLP-1 fusion proteins were genetically engineered: GLP-1 human serum albumin fusion (GLP-1/HSA), AGLP-1/HSA which has an additional alanine at the N-terminus of GLP-1, and AGLP-1-L/HSA, in which a peptide linker is inserted between AGLP-1 and HSA. Recombinant fusion proteins secreted from the Chinese Hamster Ovary-K1 (CHO-K1) cell line were purified with high purity (>96%). AGLP-1 fusion protein was resistant against the dipeptidyl peptidase-IV (DPP-IV). The fusion proteins activated cAMP-mediated signaling in rat insulinoma INS-1 cells. Furthermore, a C57BL/6N mice pharmacodynamics study exhibited that AGLP-1-L/HSA effectively reduced blood glucose level compared to AGLP-1/HSA.
Subject(s)
Glucagon-Like Peptide 1/metabolism , Serum Albumin/metabolism , Alanine/genetics , Alanine/metabolism , Animals , Blood Glucose/analysis , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Dipeptidyl Peptidase 4/metabolism , Glucagon-Like Peptide 1/genetics , Glucose Tolerance Test , Half-Life , Humans , Hypoglycemic Agents/metabolism , Hypoglycemic Agents/pharmacokinetics , Mice , Mice, Inbred C57BL , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacokinetics , Serum Albumin/genetics , TransfectionABSTRACT
A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79 % identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified by using Ni-NTA affinity chromatography and characterized. The optimum temperature and pH for DERA were 50 degrees C and 6.0, respectively. The specific activity for deoxyribose 5-phosphate (DR5P), substrate, was 62 micronmol/min/mg. The Km value for DR5P was determined to be 145 mM with the Kcat value of 3.2 times 10(2 /sec) from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).
Subject(s)
Aldehyde-Lyases/chemistry , Bacterial Proteins/chemistry , Gene Expression , Paenibacillus/enzymology , Aldehyde-Lyases/genetics , Aldehyde-Lyases/isolation & purification , Aldehyde-Lyases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cloning, Molecular , Enzyme Stability , Kinetics , Molecular Sequence Data , Paenibacillus/classification , Paenibacillus/genetics , Paenibacillus/isolation & purification , Phylogeny , Sequence Homology, Amino Acid , Soil MicrobiologyABSTRACT
A gene encoding glucansucrase was identified in Leuconostoc lactis EG001 isolated from lactic acid bacteria (LAB) in Kimchi, a traditional Korean fermented food. The L. lactis EG001 glucansucrase gene consists of 4503 bp open reading frame (ORF) and encodes an enzyme of 1500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (75%) to that of dextransucrase of L. mesenteroides. The gene was cloned and over-expressed in Escherichia coli strain. The recombinant enzyme was purified via Ni-NTA affinity chromatography and its enzymatic properties were characterized. The enzyme exhibited optimum activity at 30 degrees C and pH 5.0. In addition, the enzyme was able to catalyze the glycosylation of l-ascorbic acid to l-ascorbic acid 2-glucoside. The glycosylated product via EG001 glucansucrase has the potential as an antioxidant in industrial applications.
Subject(s)
Glycosyltransferases/metabolism , Leuconostoc/enzymology , Amino Acid Sequence , Base Sequence , Glycosyltransferases/genetics , Glycosyltransferases/isolation & purification , Leuconostoc/classification , Leuconostoc/genetics , Molecular Sequence Data , PhylogenyABSTRACT
A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific activity was 137 micromol/min/mg. The Michaelis constant (k(m) value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 degrees C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.
