The Construction And Expression Of The PHLD Fusion Protein In Pichia Pastoris And Its Activity Evaluation

Sleep is a very important physiological process of human life-sustaining. However, dueto the increasing competition, insomnia has become a very common phenomenon. Therefore,the development of an effective and safe anti-insomnia drug has become an urgent medicaland social problem. Delta-Sleep-Inducing Peptide (referred DSIP) is a nine-peptides thatcould promote sleep through the induction of slow wave sleep. DSIP has been the focus ofdomestic and foreign scholars because of its strong the activity and few toxic side effects onthe human body even with minimal concentration.However, with smaller molecular weight,the metabolic half-life of DSIP is shorter, all that greatly limits its practical application inclinical. The key to solve the above problem is to prolong the half-life of DSIP, so in thisproject we fused the PTD, HSA and DSIP with a linker, in order to develop a new, potentialmore efficient anti-insomnia drug.In this article, the Pichia pastoris preferred codons were selected and thePTD-HSA-Linker-DSIP (referred PHLD) gene was obtained with PCR technology. After PCRidentification, restriction endonuclease identification and gene sequencing showed thatpPIC9k/PHLD expression vector was constructed successfully. Recombinant plasmidpPIC9K/PHLD was transferred into his-strain GS115, and then was screened with highresistance to G418(2mg/mL in YPD) to obtain the strain with high expression level. Finnally,it was induced with methanol, then the engineering strain expressed the fusion protein in theyeast cell and the the growth of the fusion protein and the preparation conditions wereexplored primarily：The YPD medium was selected and shaking cultured for16-18hours. Thecultures were inoculated the inoculum size by10%to the BSM medium and cultured for48hours, then adding methanol to induce the expression of fusion protein. Adding methanol to afinal concentration of1%every24hours and after5days the supernatant was collected.Finally, pure samples were obtained by hydrophobic interaction chromatography separationand G25desalting.In this article, PHLD fusion protein was expressed highly in the yeast, and the highpurity interest protein was obtained by Butyl Sepharose4Fast Flow hydrophobic interactionchromatography and G25gel chromatograpgy desalting. Then use the classic pentobarbitalsleep experiment to evaluate the activity of PHLD fusion protein, the experimental resultsshow that the PHLD fusion protein could extend the sleeping time in mice, which indicatethat the fusion protein retained the activity of Delta-Sleep-Inducing Peptide.