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Liu H.,East China University of Science and Technology | Liu H.,Shandong Dong E E Jiao Co. | Liu H.,Shandong Ehua Biopharmaceutical Co. | Zhou X.,East China University of Science and Technology | And 5 more authors.
Biotechnology and Applied Biochemistry | Year: 2014

Insulin precursor fusion protein expressed in Pichia pastoris is a single-chain protein with a spacer peptide (EEAEAEAEPK) localized at its N-terminal. Currently, the one-step transpeptidation reaction with low yield and high cost is generally employed to convert the insulin precursor fusion protein into human insulin ester. In this study, a two-step transpeptidation reaction was proposed separating the cleavage step from the coupling step so that each reaction was performed under its optimal conditions. Using this method, the total efficiency doubled and the reaction time was shortened compared with the one-step method. In addition, the amount of O-t-butyl-l-threonine t-butyl ester and trypsin dosages were reduced by 50% and 75%, respectively. This two-step transpeptidation strategy was simple and efficient and could be used for the pharmaceutical production of human insulin. © 2013 International Union of Biochemistry and Molecular Biology, Inc. Source

Ma Y.-Z.,Peking Union Medical College | Li L.,Peking Union Medical College | Song J.-K.,Peking Union Medical College | Niu Z.-R.,Peking Union Medical College | And 5 more authors.
Journal of the Neurological Sciences | Year: 2015

Stroke is a major cause of death and disability worldwide. However, treatment options to date are very limited. To meet the need for validating the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental stroke models were critical for preclinical research. However, commonly used embolic stroke models are reluctant to mimic the clinical situation and not suitable for thrombolytic timing studies. In this paper, we established a standard method for producing a rat embolic stroke model with autologous thrombus formed within the common carotid artery (CCA) by constant galvanic stimulation. Then the thrombus was shattered and channeled into the origin of the MCA and small (lacunar) artery. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured at 2, 4 and 6 h postischemia. This model developed a predictable infarct volume (38.37 ± 2.88%) and gradually reduced blood flow (20% of preischemic baselines) within the middle cerebral artery (MCA) territory. The thrombus occluded in the MCA was able to be lysed by a tissue-type plasminogen activator (t-PA) within 4 h postischemia. The techniques presented in this paper would help investigators to overcome technical problems for stroke research. © 2015 Elsevier B.V. Source

Liu H.-F.,East China University of Science and Technology | Liu H.-F.,Shandong Ehua Biopharmaceutical Co. | Liu H.-F.,Shandong Dong E E Jiao Co. | Zhou X.-S.,East China University of Science and Technology | And 4 more authors.
Huadong Ligong Daxue Xuebao/Journal of East China University of Science and Technology | Year: 2013

Aiming to improve the fermention yield of insulin precursor (IP) in Pichia pastoris, a spacer peptide of 10 amino acids (EEAEAEAEPK) was introduced into the N-terminal of the IP. This sequence might cause the possibility of the N-terminal heterogeneity of the products. The IP produced in Pichia pastoris was purified by ion-exchange chromatography and reversed phase chromatography. The purified samples were analyzed with MALDI-TOF-MS and N-terminal sequencing. Results proved the existence of N-terminal heterogeneity and helped to find the possible causes for this phenomenon. The results also indicated that the IP produced by P. pastoris could generate a mutated insulin with deletion of threonineB30 (desB30) in chain B which posessed the characteristics of N-terminal homogeneity and no degradation in C-terminal after digested by trypsin. This research provided a new method for human insulin and detemir insulin production. Source

Liu H.,East China University of Science and Technology | Liu H.,Shandong Dong E E Jiao Co. | Liu H.,Shandong Ehua Biopharmaceutical Co. | Zhou X.,East China University of Science and Technology | And 5 more authors.
Process Biochemistry | Year: 2013

The insulin precursor (IP) expressed in Pichia pastoris is a single-chain peptide fused with a spacer peptide (EEAEAEAEPK) localized at its N-terminus and containing three trypsin cleavage sites in the polypeptide chain. The IP fusion protein is trypsinized to generate the insulin product desB30, which has a deletion of threonineB30. The three restriction sites on IP fusion protein had different affinities for trypsin and were digested in sequential order. Further analysis showed that approximately 20% of the IP digestion intermediates could not be converted into the final desB30 product if the IP fusion protein was digested in an aqueous phase. This result can be attributed to the formation of IP dimers or hexamers, which could restrict enzyme reactivity in the aqueous phase. To enhance the conversion yield of the IP fusion protein to desB30 products, a new digestion method was established. The IP was digested in the eluent that resulted from reverse phase chromatography during the purification process, which improved the yield of digestion from 80.2% to 95.6%. © 2013 Elsevier Ltd. All rights reserved. Source

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