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Expression of Tryoptophan Hydroxylase-2 in E.Coli and Construction of Expression Vector in Pichia Pastoris Expression System

Author: ChenJingShun
Tutor: YuanQiPeng
School: Beijing University of Chemical Technology
Course: Chemical Engineering and Technology
Keywords: 5 - Hydroxytryptophan Tryptophan hydroxylase -2 E. coli Pichia pastoris Expression
CLC: TQ925
Type: Master's thesis
Year: 2011
Downloads: 53
Quote: 0
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Abstract


5 - hydroxy-tryptophan (5-HTP) is an important inhibitory neurotransmitter precursors of 5 - hydroxytryptamine (5-HT), it is the brain that controls emotions, behavior, sleep, appetite chemicals. 5-HTP is not only successful antidepressant, used in the treatment of insomnia, can also be effective in helping to produce satiety, thereby reducing appetite, has a certain anti-obesity. Tryptophan hydroxylase the -2 (TPH-2) is the rate-limiting enzyme in the synthesis of 5-HT the direct catalytic tryptophan 5-HTP is generated. This study is the first to be obtained through the expression of exogenous TPH-2 protein has the important academic value and may be an important way for the industrial production of the new 5-HTP. This study focuses derived from human TPH-2 gene expression in E. coli expression system and Expression Vector Construction in Pichia pastoris expression system integration. Study constructed prokaryotic expression vector pET32a-TPH pGEX-4T-1-TPH and Pichia pastoris expression vector pPICZa-TPH, and pPIC3.5k-TPH, respectively, in E. coli BL21 (DE3) expression system and Pichia expression system for recombinant protein expression studies. In the E. coli expression system, by two vectors were successfully first expression having a His-tagged fusion protein and fusion protein with Gst tagged. Pichia expression system, the first successful target gene integrated into the genome of Pichia pastoris, build a stable exogenous expression of the engineered bacteria. In Small Things convert tryptophan to produce 5-HTP, screening 40 fungal dozen strains of actinomycetes and unknown isolated and purified from the Ghana seed strains. By first batch culture, the transformation method in the growth of the microorganism added to the logarithmic phase substrate, the substrate conversion, as well as to prevent the use of the microorganism itself metabolic system on the substrate tryptophan, by using the method of resting cells transformed microbial conversion. Such a method can reduce consumed tryptophan own metabolism caused by microorganisms. Microorganisms soon after culture was centrifuged to obtain bacterial cells, the bacterial cells dispersed in a physiological saline containing the substrate for the conversion of the substrate. The two microbial to convert tryptophan generation 5-HTP study explored the microbial conversion of tryptophan production capacity of 5-HTP, to carry out the production of 5-HTP biotransformation laid the foundation for future generations. In order to thoroughly study the physiological function of 5-HTP, carried out by experiments in mice 5-HTP suppress appetite, help the study of the function of weight loss. By comparison with the positive control and blank control, the mice twice a day, one hour before feeding single gavaged 0.5mL 0.1mg/mL concentration of the 5-HTP, after period of 7d feeding, discovered through observation and statistical with mice given normal saline weight increase of 7.4% compared to the body weight of mice gavage 5-HTP overall decline of 2.5%. Prove the efficacy of 5-HTP has some appetite suppressants to help lose weight.

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