Fusion Protein Synthesis and Purification. question. Answer all question below r
ID: 164305 • Letter: F
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Fusion Protein Synthesis and Purification. question. Answer all question below regarding this question in image . Thank you.
1. Why did the authors use BL21(DE3) gold cells, and not wild type E. coli?_______________
2. What is the name of the protein being purified in this method?______________________
3.At what absorbance (OD600) did the authors induce the cells? Why at this absorbance?_______________________
4.How do you prepare the 20 ml of STE buffer the authors used to dissolve the cells (show work)?____________________
TRIS______________?
NACL_________________?
EDTA_____________________?
5- What is the purpose of EDTA in this buffer?__________________________
6-What three different techniques did the authors use to lyse these bacterial cells?________________________
7- What is DNase I? Why is it listed in units/ml and not mg/ml?________________________--
8-What type of chromatography was used to purify the recombinant proteins?________________________
9-What was used to elute the protein from the beads?_____________________
10-What technique did the authors use to concentrate the protein? _______________________________
How did they determine the protein’s concentration?__________________________________
11-Why was propylene glycol added to the protein after concentration?_______________________
Fusion Protein Synthesis and Purification Single colonies of Escherichia coli XL1 blue or BL21(DE3) gold (Stratagene) containing plasmids with inserts encoding GST-tagged ARD1 or related proteins were added to 5 ml of Luria-Bertani broth containing ampicillin at 100 ug/ml. After incubation overnight at 37°C with shaking, the culture was added to 500 ml of the same medium and incubated at 37 Cwith shaking until A600 30.6. lsopropyl-B-D-thiogalactoside was added (0.2 mM final concentration), and after incubation at 37 Cfor 4 h, cells were sedimented by centrifugation (5,000 X g for 10 min) and stored at -20 C. Recombinant proteins were purified essentially as described by Frangioni and Neel (20) Briefly, cells were dispersed in 20 ml of STE buffer (10 mM Tris-HCI, 150 mM NaCl, 1 mMEDTA, pH 8) containing lysozyme at 1mg/ml. After incubation for 1 h on ice and the addition of 1% Triton X-100, cells were sonified and incubated (1 h at room temperature, shaking) with DNase l (Roche Applied Science) at 44 units/ml. Inclusion bodies were collected by centrifugation (15,000 X g for 15 min) and dispersed in 20 ml of STE. After the addition of 1 ml of 20% Sarkosyl and intermittent mixing (vortex mixer, 5 s every 30 s) for 5-10 min,1 ml of 20% Triton X-100 was added, insoluble material was discarded after centrifugation (15,000X g for 10 min at 4 C), and the clear supernatant was incubated (2 h at 4°C) with 0.25 ml of reduced glutathione-Sepharose. The mixture was transferred to a column, and beads were washed three times with 10 ml of STE buffer. Bound proteins were eluted with three 0.5-ml portions of 10 mM reduced glutathione in 50 mM Tris/HCl (pH 8) and concentrated by using Microcon centrifugal filter devices (10,000 or 100,000 molecular weight cut-off; Millipore). The protein concentration was determined by the Bradford method (21). Purity assessed by silver staining after SDS/PAGE was >90%. After addition of propylene glycol (35% final concentration), protein (0.1-1 mg/ml) was stored in small portions at -20°C. For ubiquitinylation assays, two different preparations of GST- or His-ARD1 protein were usedExplanation / Answer
1.BL21(DE3) gold cells are useful for protein expression at higher level.
2. ARD1 or ADP-ribosylation factor domain protein 1
3. The author induce the cell until the absorbance value at 600nm reached the value of 0.6. When the growth of the bacteria increases, the optical density also get increased. There is a chance that some of the wavelegth get scattered due to the presence of nuclic acids or proteins in the culture medium. However, OD of 600 generally seems to give proportional absorbance value with bacterial growth.
4. lets assume T the stock concentraiton of Tris-HCl = 1M or 1000mM
Nacl=1M or 1000mM
EDTA= 0.5M or 500mM
Thus for 20ml STE buffer we require ( 1000/10=100 fold diluted Tris-HCl) or 20X 1000/ 100 = 200?l Tris-HCl
(1000/150=6,6 fold diluted NaCl)or 20X 1000/ 6.6 = 3000?l or 3ml Nacl
(500/1= 500 fold diluted EDTA) or 20 X 1000/ 500= 40 ?l EDTA
Thus we need, 3ml of NaCl (1M), 200?l Tris-HCl( 1M), 40?l EDTA (0.5M) and 16.76ml water.
5. In lysis buffer EDTA controls osmolarity and acidity of lysate. Additionally EDTA prevent nucleic acid degradation by enzymes.
6. Addition of lysis buffer
Sonication
Centrifugation
7. DNase I is an endonuclease and useful for claving out DNA which is not required to be present in the lysate. DNase I is expressed as unit /ml as 1 unit means this is the amount of enzyme which is essential to produce an increment of absorbance of 260nm ( which is used for DNA ) to the level of 0.001 per min per mL at normal room temperature for polymerized DNA. Or in other words this much enzyme is required for breaking the phosphodiester bonds of the nucleotides.
8. Affinity chromatography.
9. Glutathione
10. centrifugal filtration device
11. propylene glycol is used in the purified protein so that the protein remains stable during storage.
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