Literature Report(生物专业原版全英文文献汇报)

Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
1.Introduction/ Progress
(4) However, within the last couple of years, plant proteins have successfully been expressed in P. pastoris at high levels (Fierenset al., 2004; Yoneyama et al., 2007; Pan et al., 2007). (5) In this study we present successful high-level expression of the Hordeum vulgare α-amylase/subtilisin inhibitor (BASI) in P.pastoris. (From Journal of Biotechnology , 2008)
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.8. Protein purification
(1) BASI was precipitated from the supernatant by adding solid ammonium sulphate ((NH4)2SO4) to 3.2 M final concentration, and the solution was stirred for 30 min. (2) BASI containing fractions from the column were analysed by SDS-PAGE and pure fractions were pooled as the product of the purification. Back
2. A Manual for the Preparation and Transformation of Pichia pastoris Spheroplasts
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
College of life science
1.Introduction/ Progress
(7) Compared to previous attempts to express BASI in P. pastoris (Bønsager et al., 2003), various conditions have been optimized in this study, including signal sequence, codon usage, transformation technique and growth conditions.
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.4. Transformation
(1) Prior to transformation all plasmids were linearized by BglII digestion, following gel purification using MinElute QIAGEN (Hilden, Germany). (2) Transformations were done by electroporation using a Gene Pulser (BIO-RAD, USA), voltage: 1500 V, capacitance: 25μF and resistance: 200 Ω.
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
(3) To induce expression P. pastoris was grown in 500 mL BMSY media at 28◦C, 280 rpm. After 24h growth, expression was induced by adding 50% methanol to a concentration of 0.5% (v/v). To maintain induction 50% methanol was added to 0.5% (v/v) every 24 h.
(3)Pichia pastoris is one of the eukaryotic expression hosts that have been developed into a highly successful expression system (Macauley-Patrick et al., 2005).
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.3. Preparation of competent cells
(1) Conduct the step of this experiment according to the procedure of this paper. (2)The competent cells were either used right away, or stored at −80◦C for later use.
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.6. Insoluble blue starch assay
(1) AMY2 (3.75 nM) was preincubated with BASI at various concentrations (ranging from 0 to 50 nM) in a total volume of 400μL for 10 min at 37◦C in reaction buffer.
Hubei Normal University
Literature reFra Baidu bibliotekort
Reporter :Xiangru
Chen
Key laboratory of structure and function of protein
College of life science
Content
1. High-level expression of the native barley α-amylase/subtilisin inhibitor in Pichia pastoris
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.7. Southern blotting
(1)Total DNA was purified by using the MasterPure TM Yeast DNA Purification kit from Epicentre (Madison, USA) (2) The quality and concentration of the DNA was determined by agarose gel electrophoresis and spectrophotometry.
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.5. Screen for single-copy transformants
(1)Single colonies were picked from the initial YNB plates and resuspended in 10μL water. (2) Cells were lysed by microwaving for 1 min.
High-level expression of the native barley α-amylase/subtilisin inhibitor in Pichia pastoris
Content
1.
Introduction / Progress
2. 3.
Materials and methods
Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.2. Cloning
(1) A new polylinker was introduced into pPIC9K by replacing the BamH I– Not I fragment, including the alpha mating factor secretion signal, creating pPIC9Kp (Fig. 1).
Results
4.
Discussion
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Copyright © 2014 Xiangru Chen , All rights reserved.
1.Introduction/ Progress
(1)Previously most plant proteins expressed in P.pastoris resulted in low yields (Cereghino and Cregg, 2000). (2) BASI(the Hordeum vulgare α-amylase/subtilisin inhibitor ) has previously been expressed in P. pastoris , but yields were very low (Bønsager et al., 2003).
(6) In this paper a high-level expression system for the bifunctional α-amylase/subtilisin inhibitor from barley is presented.
Copyright © 2014 Xiangru Chen , All rights reserved.
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Copyright © 2014 Xiangru Chen , All rights reserved.
College of life science
2. Materials and methods
2.1. Strains, culture media and growth conditions
(1)P. pastoris(Invitrogen, USA) GS115 was used for heterologous expression of BASI. (2) YPD medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose) YNB (Yeast Nitrogen Base with ammonium sulphate without amino acids) BMSY media