Effect Of Signal Peptide On The Secretory Expression Of Recombinational Protein In Yeast Expression System

In the yeast secretory expression system, cis-elements containing promoter, signal peptide, terminer are efficient for gene expression. In order to study the effect of signal peptide on secretory expression of recombinational protein in yeast, a mutated signal peptide (named as SI) was obtained by PCR mutagenesis from natural Inulinase signal peptide (CSS). CSS, SI and a-factor were separately cloned to a Kluyveromyces secretion expression vector, Saccharomyces cerevisiae secretion expression vector and Pichia pastoris secretion expression vector and then transformed to their corresponding hosts, in which Inulinase was used as reporter gene. We obtained the following recombinants:Y179U/pUKD-S-P-CSS-Inu-T, Y179U/pUKD-S-P-SI-Inu-T, Y179U/pUKD-S-P-aF-Inu-T; Y166/pUKD-S-P-CSS-Inu-T, Y166/pUKD-S-P-SI-Inu-T, Y166/pUKD-S-P-αF-Inu-T; GS115/pPIC9K-CSS-Inu, GS115/pIC9K-SI-Inu, GS115/pPIC9K-aF-Inu; and SMD1168/pPIC9K-CSS-Inu, SMD1168/pIC9K-SI-Inu, SMD1168/pPIC9K-aF-Inuo Detection of secretory expression level of Inulinase in the above recombinants showed that three kinds of signal peptide can effectively mediated the secretory expression of the reporter gene Inulinase in all detected yeast expression system. However, CSS and SI are more efficient than a-factor, and SI is the most efficient in guiding the secretory expression of foreign protein in all tested yeast systems. Meanwhile, a recombinant mutant GS115/pPIC9K-aF-Inu-M was obtained accidentally, in which the secreted Inulinase has smaller molecular weight than that in normal recombinant strain. DNA sequencing and enzymatic analysis of Inulinase showed that the Inulinase expressed in the mutant is the same as that in the normal strain. N-glycosylase digestion and crystallization suggest that the smaller molecular weight of Inulinase is probably due to the defect of glycosylation in the mutant. In Saccharomyces cerevisiae Haplp is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. For example S. cerevisiae is one kind of fermetative metabolism dominate yeast, while Kluyveromyces lactis is an respiratory metabolism dominate yeast specie which shows different oxygen responses.It is suspected that differences in the interaction of Haplp with its target genes may explain some of the species-related variation in oxygen responses. We examined the role of the HAP1-equivalent gene (KIHAP1) in K. lactis. KlHaplp showed a number of sequence features and some gene targets (such as KICYC1) in common with its S. cerevisiae counterpart, and KIHAP1 was capable of complementing the hap1 mutation. However, the KIHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature,28℃, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHaplp repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The Klhapl mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHaplp in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis. In Saccharomyces cerevisiae, HAP1 mainly inhibited the downstream anaerobic genes through ROXlp in aerobic conditions. To detect the mechanism that KIHAP1 regulated glucose transporter Rag1g, the equivalent gene of ROX1 in K. lactis has been found. And further research found the the pututive KIROX1 didn't respress downstream anaerobic genes and there is also no inhibitory effect on RAG1. In K.lactis KlHaplp might negatively control Raglp in other ways which haven't been found now.