ABSTRACT
Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.
Subject(s)
6-Phytase/genetics , Pichia/genetics , Plasmids , Recombinant Proteins/genetics , SaccharomycetalesABSTRACT
Objective To investigate the clinical efficacy of prucalopride in the treatment of severe chronic constipation.Methods The clinical data of 60 patients with severe chronic constipation [slow transit constipation (STC), functional defecation disorder (FDD) and constipation-predominant irritable bowel syndrome (IBS-C)] who were admitted to the Third Affiliated Hospital of Nanjing University of Chinese Medicine from February to August 2014 were prospectively analyzed.A prospective, clinical observational study was performed.Treatment plans included that patients withdrew the initial treatments of laxative and exema and took orally 2 mg prucalopride once daily for 2 weeks, and continued to be treated by oral prucalopride if frequency of the spontaneous complete bowel movement (SCBM) per week was satisfactory (or improvement of symptoms was more than 50％) till 4 weeks, and then were followed up after stopping prucalopride.If improvement of symptoms was less than 50％ after 2-week treatment, other treatment plans were performed according to symptoms of patients from week 3 to week 6 : (1) for patients with STC, prucalopride + two chain bacillus subtilis probiotic capsules were administered orally if patients were satisfied with frequency of SCBM per week and without improvement of abdominal distension;prucalopride + Chinese herb decoction were administered orally if patients had improvement of frequency of SCBM per week with abdominal distension or poor stool output;oral prucalopride + acupuncture were administered if patients were unsatisfied with frequency of SCBM per week or less bowel movements and without improvement of abdominal distension or poor stool output.(2) For patients with FDD, oral prucalopride + acupuncture + biofeed-back therapy were administered.(3) For patients with IBS-C, prucalopride + two chain bacillus subtilis probiotic capsules were administered orally if patients had abdominal distension;prucalopride Chinese herb decoction were administered orally if patients had improvement of frequency of SCBM per week and no improvement of abdominal distension or poor stool output.All patients used a diary for recording the frequency of SCBM per week, stool consistence, exertion in defecation and adverse reactions, which was submitted to doctors for inputting data at the return visit weekly.Results There was good overall medicine compliance in patients.Of 60 patients, 43 patients completed treatments (21 with STC, 11 with FDD and 11 with IBS-C).After 2-week treatment, there were 19 patients with satisfied therapeutic effects, 14 with improvement of constipation and 10 with poor therapeutic effects.After 4-week treatment, constipation in 17 patients was cured, constipation in 18 patients was improved,and constipation in 8 patients was not improved.Nineteen of 60 patients were complicated with adverse reactions within 1 week of the medication, including 6 patients dropping out of the trial due to medication withdrawal and others with improvement by symptomatic treatment or spontaneous remission.Conclusions Prucalopride is effective for the treatment of severe chronic constipation with a good toleration, and it can improve the overall satisfaction of patients combined with Chinese herb decoction and acupuncture.
ABSTRACT
This study is to evaluate the therapeutic effect of fibroblast growth factor 21 (FGF21) on type 2 diabetic mice model and to provide mechanistic insights into its therapeutic effect. Type 2 diabetic animal model was established with high calorie fat diet and low dose streptozotocin (STZ) injection. Mice were then randomized into 5 groups: model control, FGF21 0.25 and 0.05 μmol x kg(-1) x d(-1) groups, insulin treatment group. Ten age-matched normal KM mouse administered with saline were used as normal controls. Serum glucose, insulin, lipid products and the change of serum and liver tissue inflammation factor levels between five groups of mouse were determined. The results showed that blood glucose, insulin, free fatty acids (FFAs), triglycerides, and inflammatory factor average FGF-21 of type 2 diabetes model group and normal control group were significantly higher (P < 0.01), while compared with insulin group, no difference was significant. Average blood glucose, insulin, blood lipid and inflammatory factor of FGF-21 treatment group compared with type 2 diabetes group was significantly lower (P < 0.01) and insulin group has no difference with the model control group. The results of OGTT and HOMA-IR showed that insulin resistance state was significantly relieved in a dose-dependent manner. Thus, this study demonstrates that FGF-21 significantly remits type 2 diabetic mice model's insulin resistance state and participates in the regulation of inflammatory factor levels and type 2 diabetes metabolic disorders.