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 diagnosis and differential diagnosis of primary hepatic malignant fibrous histiocytoma (MFH) by ultrasound. Methods A retrospective analysis of clinical data and ultrasonic findings was performed on 16 patients with pathologically proven hepatic MFH from July 2005 to August 2012 in Eastern Hepatobilary Surgery Hospital of the Second Military Medical University. Results Of the 16 patients, all the tumors were single, which including 9 tumors located in the right lobe, 6 tumors located in the left lobe, and 1 tumor located in caudate lobe of the liver. The tumor sizes were larger than 10.0 cm in 3 cases, between 5.0 cm and 10.0 cm in 10 cases, and less than 5.0 cm in 3 cases. Tumor internal echo was heterogeneous, including 1 case with honeycomb pattern and 2 cases with mosaic sign. Most tumors were unclearly-deifned (11 cases) without obvious halo. The pathologic results identiifed 15 cases of primary hepatic MFH and 1 case of primary intrahepatic bile duct malignant ifbrous histiocytoma. Conclusions It is dififcult to make correct clinical diagnosis of hepatic MFH. A comprehensive analysis of ultrasound ifndings and clinical information is needed to obtain an accurat diagnosis of malignant liver tumors.