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Tang W.,Hubei University | Li Z.,Hubei University | Li C.,Hubei University | Yu X.,Peking University | And 4 more authors.
Protein Expression and Purification | Year: 2016

Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B. subtilis strains 168 and zj016. The yield (3.81 g/l) and specific activity (788 U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83 mg of the purified enzyme (4089.72 U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B. subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B. subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B. subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B. subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases. © 2016 Elsevier Inc.


PubMed | Hubei University, Peking University and Wuhan Institute of Biological Product Co.
Type: | Journal: Protein expression and purification | Year: 2016

Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B.subtilis strains 168 and zj016. The yield (3.81g/l) and specific activity (788U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83mg of the purified enzyme (4089.72U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B.subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B.subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B.subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B.subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases.


Yang B.-L.,Wuhan Institute of Biological Product Co. | Wang H.-Q.,Wuhan Institute of Biological Product Co. | Wang K.,Wuhan Institute of Biological Product Co. | Wang X.-D.,Wuhan Institute of Biological Product Co. | Li Y.-H.,Wuhan Institute of Biological Product Co.
Chinese Journal of Biologicals | Year: 2014

Objective To analyze the stability of live attenuated Japanese encephalitis (JE) vaccine. Methods A total of 28 batches of live attenuated JE vaccine were randomly chosen and tested for neurovirulence reversion in suckling mice and intracerebral virulence in mice according to the requirements in Chinese Pharmaceupia (Volume III , 2010 edition) and the SOP of Neurovirulecne Reversion Test in Suckling Mice and Intracerebral Virulence Test in Mice of Live Attenuated Japanese Encephalitis Vaccine maunufactured by Wuhan Insitute of Biological Products Co., Ltd. Meanwhile, the brain tissue suspension of pathogenic suckling mice was determined for vims titer. The Kendall correlation of determination results of vims titers of 28 batches of vaccine and brain tissue supernatant of pathogenic suckling mice as well as intracerebral virulence of intracerebral vims of pathogenic suckling mice to mice was analyzed. Results The vims titers of 28 batches of live attenuated JE vaccine were 6. 0 7. 1 I-gPFU/ml, while the intracerebral virulences (LD50) of intracerebral vims of pathogenic suckling mice to mice were less than 3. 0 LgLD50/0. 03 ml, and the vims titers of brain tissue supernatant of pathogenic suckling mice were 5. 8 7. 0 I-gPFU / ml. The intracerebral virulence of intracerebral vims of pathogenic suckling mice to mice showed no correlation to the vaccine vims titer or the titer of brain tissue supernatant of pathogenic suckling mice. However, vaccine vims ttier showed low correlation to the titer of brain tissue supernatant of pathogenic suckling mice. Conclusion Live attenuated Japanese encephalitis vaccine showed stable virulence. Neurovirulence reversion test in suckling mice and its judgment standard were scientific and strict, and was feasible to the quality control of vaccine. The study further indicated the higli genetic stability of strain SA14-14-2.

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