A novel strategy to enhance the expression efficiency of cloned target gene in Escherichia coli was developed. The whole expression cartridge , consisting of promoter. SD sequence , target gene and transcription termi...A novel strategy to enhance the expression efficiency of cloned target gene in Escherichia coli was developed. The whole expression cartridge , consisting of promoter. SD sequence , target gene and transcription terminator, was tandem repeatedly engineered into a expression plasmid. Consequently, the copy number of specific gene was increased substantially, leading to the improvement of expression efficiency.Using this approach, a recombinant plasmid , designed as PLYD, was constructed and transformated into the Escherichia coli strain DH5α. Upon induction , the desired protein was synthesized in a considerable level and accumulated up to 63% of the total cell proteins. The present study revealed that tandem repeating of expression cartridge provided a convenient means to improve expression level efficiently.展开更多
Small bioactive peptides, with diverse biological functions, have received increasing attention as physiologically beneficial substances in animal production. The main obstacle to wide application of small bioactive p...Small bioactive peptides, with diverse biological functions, have received increasing attention as physiologically beneficial substances in animal production. The main obstacle to wide application of small bioactive peptides is a lack of costeffective methods for mass production. In this study, we mass-production method for small bioactive peptides. used glycyl-glutamine （Gly-Gln） as a test case to develop a novel The oligonucleotide encoding Gly-Gln pro-peptide （Gpp） was designed and synthesized. Gpp includes 3 Gly-Gln dipeptides and 2 enzymatic sites for pepsin and trypase, allowing direct digestion and absorption of Gly-Gln in the gastrointestinal tract. The Gpp oligonucleotides were linked to generate an oligomeric oligonucleotide segment containing 12 tandem copies of Gpp. This 12Gpp segment was cloned and expressed in Escherichia coli vector pET32a. By optimizing culture conditions [0.1 mmol L^-1 isopropyl-β-D- thiogalactopyranoside （IPTG）, 50 μg mL^-1 ampicillin （Amp）, 30℃ for 12 h], the thioredoxin fusion peptides reached 40% of total bacterial protein. After purification, the fusion protein was fed to Kunming mice to determine its effect on mouse immune function. The results showed that similar to Gly-Gln dipeptide, Gpp polymer protein could significantly suppress the proliferation of T and B lymphocytes in blood and spleen, and additionally could significantly improve interleukin-2 （IL-2） and interleukin-6 （IL-6） secretion of blood and spleen lymphocytes. These effects were not observed in mice fed a 2 amino acids mix （glycine and glutamine）. These evidences indicated that an efficient digestion of Gpp polymer protein could be achieved when ingested into the animal gut. The expression system in this study provides a potential production method for not only Gly-Gln dipeptide but also other short bioactive peptides.展开更多
Xuanwei Ham Peptide(XHP)is a bioactive compound isolated and extracted from Xuanwei ham.It enhances the oxidative defense system by down-regulating CYP2E1 expression,reducing ROS production,and by activating the Nrf2/...Xuanwei Ham Peptide(XHP)is a bioactive compound isolated and extracted from Xuanwei ham.It enhances the oxidative defense system by down-regulating CYP2E1 expression,reducing ROS production,and by activating the Nrf2/HO-1 pathway.In order to solve the problems of low yield,high cost and time-consuming separation process of active peptides from ham.In this study,we designed three peptide sequences derived from Xuanwei ham and expressed them recombinantly.The results showed that the peptides using flexible linker peptide((GGGGS)2),rigid linker peptide((EAAAK)2)linkage as well as the head-to-tail tandem linkage were successfully expressed in the E.coli expression system.Peptide monomers were released from the fusion proteins by formic acid cleavage and purified sequentially by Ni-IDA affinity chromatography,membrane filtration and RP-HPLC.The molecular weight of XHP was confirmed to be 717.35 by ESI-MS analysis.The results of the antioxidant activity of XHP showed that the hydroxyl radical scavenging rates of the three recombinant peptides were as high as 79.46%,87.42%,and 53.60%,respectively.The hydroxyl radical activity of formic acid-cleaved XHP was reduced to 35.31%,but it showed good stability in heat,pH,metal ions and gastrointestinal digestion(GID).The molecular docking results showed that the docking of XHP to Keap1 protein was evaluated by five Molecular dynamics simulation with a maximum binding energy of-8.5 kcal/mol.This study provides references and ideas for the expression of active peptides,which can be widely used in the future can be used in the field of nutraceuticals,functional foods and pharmaceuticals.展开更多
文摘A novel strategy to enhance the expression efficiency of cloned target gene in Escherichia coli was developed. The whole expression cartridge , consisting of promoter. SD sequence , target gene and transcription terminator, was tandem repeatedly engineered into a expression plasmid. Consequently, the copy number of specific gene was increased substantially, leading to the improvement of expression efficiency.Using this approach, a recombinant plasmid , designed as PLYD, was constructed and transformated into the Escherichia coli strain DH5α. Upon induction , the desired protein was synthesized in a considerable level and accumulated up to 63% of the total cell proteins. The present study revealed that tandem repeating of expression cartridge provided a convenient means to improve expression level efficiently.
基金supported by Science and Technology Plan Project of Guangdong Province(20065010089 and 2007A020100005-1)the Scienseand Technology Plan Project of Guangzhou City, the National Basic Research Program of China(2009CB941601)the Joint Funds of the Guangdong Province and National Natural Science Foundation ofChina (u0731004)
文摘Small bioactive peptides, with diverse biological functions, have received increasing attention as physiologically beneficial substances in animal production. The main obstacle to wide application of small bioactive peptides is a lack of costeffective methods for mass production. In this study, we mass-production method for small bioactive peptides. used glycyl-glutamine （Gly-Gln） as a test case to develop a novel The oligonucleotide encoding Gly-Gln pro-peptide （Gpp） was designed and synthesized. Gpp includes 3 Gly-Gln dipeptides and 2 enzymatic sites for pepsin and trypase, allowing direct digestion and absorption of Gly-Gln in the gastrointestinal tract. The Gpp oligonucleotides were linked to generate an oligomeric oligonucleotide segment containing 12 tandem copies of Gpp. This 12Gpp segment was cloned and expressed in Escherichia coli vector pET32a. By optimizing culture conditions [0.1 mmol L^-1 isopropyl-β-D- thiogalactopyranoside （IPTG）, 50 μg mL^-1 ampicillin （Amp）, 30℃ for 12 h], the thioredoxin fusion peptides reached 40% of total bacterial protein. After purification, the fusion protein was fed to Kunming mice to determine its effect on mouse immune function. The results showed that similar to Gly-Gln dipeptide, Gpp polymer protein could significantly suppress the proliferation of T and B lymphocytes in blood and spleen, and additionally could significantly improve interleukin-2 （IL-2） and interleukin-6 （IL-6） secretion of blood and spleen lymphocytes. These effects were not observed in mice fed a 2 amino acids mix （glycine and glutamine）. These evidences indicated that an efficient digestion of Gpp polymer protein could be achieved when ingested into the animal gut. The expression system in this study provides a potential production method for not only Gly-Gln dipeptide but also other short bioactive peptides.
基金supported by the Major Science and Technology Program of Anhui,China(2021d06050001)the Anhui Provincial Natural Science Foundation of China(2108085QC148)the Fundamental Research Funds for the Central Universities of China(JZ2021HGQA0242).
文摘Xuanwei Ham Peptide(XHP)is a bioactive compound isolated and extracted from Xuanwei ham.It enhances the oxidative defense system by down-regulating CYP2E1 expression,reducing ROS production,and by activating the Nrf2/HO-1 pathway.In order to solve the problems of low yield,high cost and time-consuming separation process of active peptides from ham.In this study,we designed three peptide sequences derived from Xuanwei ham and expressed them recombinantly.The results showed that the peptides using flexible linker peptide((GGGGS)2),rigid linker peptide((EAAAK)2)linkage as well as the head-to-tail tandem linkage were successfully expressed in the E.coli expression system.Peptide monomers were released from the fusion proteins by formic acid cleavage and purified sequentially by Ni-IDA affinity chromatography,membrane filtration and RP-HPLC.The molecular weight of XHP was confirmed to be 717.35 by ESI-MS analysis.The results of the antioxidant activity of XHP showed that the hydroxyl radical scavenging rates of the three recombinant peptides were as high as 79.46%,87.42%,and 53.60%,respectively.The hydroxyl radical activity of formic acid-cleaved XHP was reduced to 35.31%,but it showed good stability in heat,pH,metal ions and gastrointestinal digestion(GID).The molecular docking results showed that the docking of XHP to Keap1 protein was evaluated by five Molecular dynamics simulation with a maximum binding energy of-8.5 kcal/mol.This study provides references and ideas for the expression of active peptides,which can be widely used in the future can be used in the field of nutraceuticals,functional foods and pharmaceuticals.
