Study On Construction And Application Of Recombinant Saccharomyces Cerevisiae Stably Expressing Long-acting Glucagon-like Peptide-1

Currently,most peptide and protein drugs are still administered by the parenteral route because of insufficient absorption from the gastrointestinal tract.Therapeutic peptides and proteins are usually used for chronic conditions,and the use of injections during long-term treatment has obvious drawbacks.In contrast to this inconvenient and potentially problematic method of drug administration,the oral route offers the advantages of self-administration with a high degree of patient acceptability and compliance,and avoids the pain,discomfort,and possibility of infections caused by injectable dosage forms.Therefore,developing the effective and successful oral delivery system,in order to maximize the therapeutic potential of this class of agents, has been an ongoing topic in pharmaceutical research.It is an innovative way that recombinant microorganisms expressing therapeutic peptides and proteins are used as drug delivery system.Both bacteria and yeasts could be used as a new drug delivery vehicles to the gastrointestinal tract.Glucagon-like peptide-1(GLP-1),so-called incretin,is a peptide hormone secreted from gut endocrine cells.It has lots of physiological actions,including glucose-dependent insulinotropic effect,stimulation ofβ-cell proliferation and so on. Therefore,GLP-1 has great therapeutic potential to treat diabetes type 2.However, the clinical application of native GLP-1 is limited by its very short half-life in vivo due principally to its rapid degradation by dipeptidyl peptidaseⅣ(DPPⅣ) and renal clearance.Accordingly,developing long-acting therapeutic GLP-1 analogs is of considerable interest.S.cerevisiae(baker yeast) is rich in vitamins,digestive enzymes and so on.As we know,it is a long history that S.cerevisiae is used as tea or beverage in Taiwan, Hong Kong and other places.If S.cerevisiae cells could be utilized as a drug vehicle, or named sustained release "capsules",to intracellularly stably express therapeutic polypeptide drugs for diabetes type 2,we might develop its therapeutic use as functional drinks and food,and even biodrug.Thus,we can prevent and treat diabetes or other chronic diseases as we enjoy these delicious drinks or food.This strategy would be very attractive. This study aims to construct recombinant S.cerevisiae intracellularly stably expressing polypeptide for the treatment of diabetes type 2 in order to lay the foundation for the development of the therapeutic use of yeast as drinks,food or biodrug.We start with the construction of yeast integrating vector containing the backbone of the multiple plasmid pUC18.For targeted integration of foreign gene into reiterated multi-copy rDNA sequence on yeast chromosome,we firstly designed two pairs of primers to amply two rDNA fragment,AB and CD.These two fragment located in a 2.2kb recombination hot region between 25SrDNA gene and 5SrDNA gene.Two fragments of AB and CD were successively cloned into EcoRⅠ/SacⅠand PstⅠsites of pUC18.The plasmid named pUC18-Sr was obtained.Using vector pYX212 as a template,two pairs of primers,U1-U2 and T1-T2,were designed to amply auxotropic selective marker URA3 gene and expression cassette containing TPI promoter,respectively.Afterwards,amplied URA3 gene and expression cassette fragment,were ligated into SalI/XbaI and XbaI/KpnI sites of pUC18-Sr.The vector pNK1 was obtained.In addition,expression cassette fragment and selective marker Leu2-d gene fragment from pYEX were successively ligated into XbaI/KpnI and blunted XbaI sites of pUC18-Sr.Vector pNK2 was generated.To test the function of vectors pNK1 and pNK2,BamHI/HpaI fragment from plasmid pEGFP-N1 containing green fluorescent protein gene(GFP) was ligated into BamHI/XmaI site in expression cassette to construct expression vectors pNK1-GFP and pNK2-GFP.The linearized pNK1-GFP and pNK2-GFP were transformed into Saccharomycess cerevisiae.The transformants containing URA3 gene were selected on Ura selective medium.The transformants containing Leu-2d gene were selected on Leu selective medium.The results showed that the function of two selective genes is normal.After transformants were cultured overnight,green fluorescence from transforms was observed by fluorescence microscope,indicating promoter TPI might drive the expression of GFP gene.Furthermore,the results of Southern blot demonstrated that GFP gene was integrated into yeast genome.Under the non-selective condition,Above 99%transformant containing URA3 gene SG2 and 100%transforms containing Leu-2d gene still grew on Ura~- selective medium and on Leu~- selective medium,respectively,throughout sequential passage of 50 generations. The above results indicated that both pNK1 and pNK2 guarantee the mitotic stability of GFP gene integrated into yeast chromosome.Because 58%cells from transformants containing pNK1-GFP were of green fluorescence by fluorescence microscope observation in the same cell concentration,pNK1 was selected to express recombinant oral long-acting glucagons-like peptide-1(rolGLP-1).Utilizing a ten-tandem-copy rolGLP-1 fusion gene constructed in our lab as a foreign gene,the expression vector pNK-GLP was constructed by introducing rolGLP-1 fusion gene into multi-copy Yip(pNK1).The linearized pNK-GLP was transformed into protease-deficient S.cerevisiae BJ2407,and the transformants were selected on auxotrophic(Ura~-) selective plate.Three transformants were randomly selected.Then,the positive transformants were identified by Southern blot.After growth determination in YPD medium,transformant SG2 was selected and used for further research.The small-scale cultures of transformant SG2,using glass beads,was broken.The cell lysate separated on the SDS-PAGE were analyzed by West blot,an approx.36kD protein band corresponding to the predictive molecular weight was detected.The analysis result indicated that the rolGLP-1 fusion protein was intracellularly expressed in transformant SG2.After growth in YPD medium with the inoculum and medium ratio of 1:1,SG2 was disrupted.The cell lysate was applied to Ni-NTA resin column,and rolGLP-1 fusion protein was purified.The expression level of rhGLP-1 analogue fusion protein was assessed by densitometric scaning,and the amount of rolGLP-1 fusion protein evaluated by Bradford method reached 0.84mg/g wet cells.After SG2 continuously growing in non-selective medium, rolGLP-1 fusion gene was of mitotically stable,and still was expressed in SG2 by analysis of Western blot.The SG2 cells in late exponential phase were dried by freeze-drying technique,and then lyophilized SG2 cells were used for the assessment of glucose-lowering effects in hyperglycemic rats.After 10h fasting,Wistar rats were intravenously infected with 60mg streptozotocin/kg to induce the hyperglycemia.The serum glucose level of hyperglycemic rat was 25.1±3.5mM.The hyperglycemic rats were administered various doses(5g/kg,0.5g/kg,0.1g/kg) of recombinant yeast SG2,positive control group were given 0.1g/kg Metformin Hydrochloride and model group were given 5g/kg yeast BJ2407 by gastric gavages for 20 days.The analysis of in vivo activity indicated that serum glucose value of rats in model group still retained high level (26.2±2.9mM).Oral administration of recombinant yeast SG2 at 0.5g/kg/d and 0.1g/kg/d could not obviously lower serum glucose level.Taking 0.1g/kg/d Metformin Hydrochloride or 5g/kg/d SG2 significantly lowered serum glucose level. Oral administration of 0.1g/kg/d Metformin Hydrochloride decreased serum glucose level of hyperglycemic rats from 25.1±3.9 to 19.1±4.3mM(P＜0.01).Oral administration of SG2 at 5g/kg/d for 20 days also lowered serum glucose level of hyperglycemic rats from 24.8±4.0 to 21.2±3.8mM(P＜0.05).In addition,the abnormal changes of drinking amount,food intake and body weight of hyperglycemic rats also were significantly improved.In summary,two multi-copy integration vectors of pNK1 and pNK2,using strategy based on integration into the ribosomal DNA cluster,were successfully constructed in this study.They contain,except for constitutive promoter TPI, auxotrophic selective marker URA3 gene and Leu-2d gene,respectively.Whereafter, the expression vector containing ten-tandem-copy rolGLP-1 fusion gene,pNK-GLP, was constructed and transformed into S.cerevisiae BJ2407.recombinant yeast SG2 stably expressing rolGLP-1 fusion protein was obtained.Meanwhile,the result of bioactivity test indicated that oral administration of recombinant yeast SG2 could lower serum glucose level of hyperglycemic rats.It laid the well foundation for further development of the use of recombinant S.cerevisiae as beverage,food or biodrug for the prevention and treatment of diabetes type 2.