Aim: Expression and visualization of viral capsids protein.
Translation vectors are used for the expression of target genes derived from eukaryotic sources. Subcloning of protein-coding region of DNA into a pET vector is used for expression. The unique restriction sites found in the multiple cloning region of the pET vectors is used for cloning inserts in a defined orientation using two different sites. For protein production, the recombinant plasmid is transferred to host E. coli strains containing a chromosomal copy of the gene for T7 RNA polymerase. These hosts are lysogens of bacteriophage DE3, a lambda derivative that has the immunity region of phage 21 and carries a DNA fragment containing the lacI gene, the lacUV5 promoter, and the gene for T7 RNA polymerase. The only promoter known to direct transcription of the T7 RNA polymerase gene is the lacUV5 promoter, which is inducible by isopropyl--D-thiogalactopyranoside (IPTG). The promoter is followed by a unique cloning site, and some carry translation initiation signals (sl0) and a transcription terminator (TOp) or an RNase HI cleavage site. This system is becoming very popular for heterologous protein expression. Addition of IPTG to a growing culture of the lysogen induces T7 RNA polymerase, which in turn transcribes the target DNA in the plasmid. Requirements: LB media, TBM, ampicillin (100mg/ml), IPTG (1M), PBS(1X), 5% Triton X 100 (if required), Cobalt-NTA, Imidazole, autoclaved distilled water. Composition of TBM: Bactotryptone : 12 g Bacto yeast extract: 24g Glycerol: 4 ml KH2PO4: 2.31g K2HPO4: 12.54g H2O make upto 1 L Procedure: Preparation of Media and primary culture 1. Weighed 2 g of LB and dissolved in 100 ml of distilled water. The TBM media was prepared with above composition and distributed in conical flasks using only 25% capacity of the media. Autoclaved the LB and TB media.
2. Ampicillin stock of concentration 100 mg/ml was prepared in autoclaved distilled water in laminar hood and stored at 4C. 3. Ampicillin stock (100mg/ml) was added to LB medium with same volume as that of LB (l) in laminar hood to final concentration of 100 g/ml and inoculated the 5 ml culture with three- four colonies from the plates. 4. Incubated the cultures overnight at 37C on shaker with 150 rpm. Induction for protein expression: 1. Ampicillin from 100 mg/ml stock was added to the TBM media in laminar hood to have final concentration 100 g/ml. 2. Inoculum (1 %) was added to the flask containing TBM in laminar hood. 3. Incubated the culture for 2.5 hour at 37 C and checked the OD600. If the OD600 is 0.4-0.6, IPTG (from the 1 M stock) is added to each flask of culture to make final concentration of 0.3 mM and incubated for 2 days at 16C at 150 rpm. Cell harvesting and Protein extraction 1. After 2 days centrifugation of the culture is done to harvest the cells at 4oC, 7000 rpm, 10 mins. 2. The pellet is washed with PBS (1X) to remove the traces of TBM media. The pellet is completely dissolved in 1X PBS (1/10th of culture used in induction) and ultra Sonication is done at 4oC 40% amplitude on pulse: 02 sec, 05 sec off pulse for 5 mins. 3. Centrifuged the cells at 14000 rpm at 4oC for 20 mins to collect the final supernatant. 4. The final pellet was washed with PBS and centrifuged again at the 4 oC for 20 mins as to collect the supernatant and is stored at 4oC for further use. Protein purification by Affinity chromatography: Protein purification by Co-NTA column The protein of interest is isolated from complex mixture of proteins in purified form. Affinity Chromatography (Immobilized metal ion affinity chromatography) by Cobalt NTA column was performed as the protein of interest was His tagged. Cobalt has affinity for the imidazole ring of His residue hence it will be remain bound to the column and rest of the protein will come out in flow through. After the washing of the column the protein is eluted using 500 mM imidazole. Chemicals: Co-NTA column, PBS, Imidazole 20mM, 250mM.
Protocol: 1. The soluble fraction (supernatant) was collected and filtered through 45 m filters. 2. The soluble fraction was bound to 1 ml Co-NTA beads at 4C for 30 min or The flowthrough collected was allowed to pass through the bead suspension number of times (thrice). 3. The beads were washed twice with 10ml PBS, pH 7.4. 4. The non specific bound protein was removed by washing with 20 mM Imidazole. 5. The protein was eluted with 4ml of 250mM Imidazole in PBS, pH 7.4.
Protein expression table
Date
TBM Culture used
1 X PBS Sample used for Column used to Affinity dissolve the pellet Chromatography per Experiment used
PBS
PBS
Elution
wash 1 Wash2
24.1.13 06.2.13
500 ml 1L
35 ml 35
10 ml 10 ml
1.5 ml 1 ml
10 ml 10 ml
10 ml 10 ml
4ml 4ml
Protein purification table: Fraction Volume (ml) Protein concentration (mg/ml) F1 F2 45 52 Total Protein (mg) F1 1125 F2 1560
Supernatant
CCMV CCMV F1 F2 27 30
Affinity 12 Chromatography
12
2.3
2.2
27.6
26.4
Protein visualization SDS-PAGE is performed of all the fractions, soluble and insoluble fraction, all the washed fractions and eluted protein.
Sample preparation: sample 5 x dye Sample (l) (l) 40 60 Water (l) Actual amount loaded ( l) 12
TCL, SF, IF, FT
100
PBSW, 40 Elution
160
32
Where; TCL = total cell lysate SF = soluble fraction IF = Insoluble fraction PBSW =PBS wash of the insoluble fraction.
CCMV F1
CCMV F2