2.

2. Construction of the TAT-CPG2 expression vectorTo evaluate if the fusionTAT segment is exposed and does not have any interference with the CPG2 domain,a 3D protein model by homology modeling using phyer2 server was created (Kelley and Sternberg,2009). For expression in E. coli, the amino acid sequence of CPG2 from PseudomonasSp. Strain RS-16 was codon-optimized by codon usage wrangler server(https://www.mrc-lmb.cam.

ac.uk/ms/methods/codon.html) and evaluated using genscript server (https://www.genscript.com/tools/rare-codon-analysis).

A TAT-CPG2 expression vector was constructedto express the basic domain (YGRKKRRQRRR)of HIV-1 TAT fused with CPG2 in frame with an N terminal 6xHis tag. The synthetic DNA sequence encoding TAT-CPG2 was subclonedinto the BamHI and NdeI sites of pET-14b. The control vector expressed CPG2, was constructedby inserting sequence encoding CPG2 coding sequence in the same sites intopET-14b without the TAT domain. Expression vectorswere transformed to into E. coli BL21 (DE3) by heat shock at 42 ?C for 1 min and chilled on ice for 2 min. 2.3.

Expression and purification of TAT-CPG2 fusionproteinsTransformed bacteria with CPG2 and TAT-CPG2 constructs weregrown in the LB medium containing 100 µg/ml ampicillin at 37 ?C to reach an optical density of0.6 at 600 nm. ExpressionM1  of the fusion proteins was induced at 0.5 mM and 1 mM of IPTG. Cellswere grown at either 37 ?C or 28 ?C for 4 h. To increase the level of soluble TAT-CPG2,the induction temperature and IPTG concentration for proteinexpression were optimized.

Experiments were carried out using0.5 and 1 mM   IPTGand the inductionprocess was performed at 28 ?C and 37 ?C. Purificationof CPG2 and TAT-CPG2 fusion proteins were carried out under native and denaturingconditions by the batch method of Qiagen. For purification of recombinantproteins under native condition the bacterial cellswere harvested by centrifugation at 10,000×g and resuspended in binding buffer(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole,and 1 mM PMSF at pH 8). Lysozyme was added at a concentration of 0.1 mg/ml andincubated on ice for 30 min.

Cells were then disrupted by sonication (6 ×10 sbursts at 200–300 W with a 10 s cooling period in-between)on ice. The bacterial lysates were centrifuged (10,000×g at 4 °C for 30 min), andthe supernatants were added to a 50% Ni-NTA resin pre-equilibrated with bindingbuffer and mixed gently by shaking (200 rpm on a rotary shaker) at 4 °C for 60min. The lysate–Ni-NTA mixture was loaded on an emptyPD10 column. The column was washed twice with 4 ml wash buffer (50 mM NaH2PO4,300 mM NaCl, 20 mM imidazole, pH 8). The CPG2 and TAT-CPG2 fusion proteins wereeluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl,250 mM imidazole, pH 8).  Eluted proteinswere desalted on a PD10 desalting column.

For purification under denaturing condition, pelletsfrom purification under native condition were resuspended in buffer B (100 mM NaH2PO4, 10 mM Tris-HCl, 8 Murea, pH 8) and stirred for 60 min at room temperature. The mixture was thencentrifuged at 10,000×g for 30 min at room temperature to pellet the cellulardebris. Supernatant was mixed with 50% Ni-NTA resin and gently shaked (200 rpmon a rotary shaker) at room temperature for 60 min. The lysate–Ni-NTA mixture wasadded to a column and washed twice with 4 ml buffer C (100 mM NaH2PO4,10 mM Tris-HCl, 8 M urea, pH 6.3). The recombinant proteins were eluted 4 timeswith 0.

5 ml buffer D (100 mM NaH2PO4, 10 mM Tris-HCl, 8 Murea, pH 5.9), followed by 4 times elution with 0.5 ml buffer E (100 mM NaH2PO4,10 mM Tris-H, 8 M urea, pH 4.5).

Monomers generally elute in buffer D, whilemultimers and aggregates elute in buffer E. The eluted proteins were desaltedby PD10 desalting column. The purified fusion proteins were verified bySDS/PAGE, coomassie brilliant blue staining and western blot analysis withanti-His6-peroxidase antibody (1:500; Roche). The protein concentrations wereestimated by the Bradford method (Bradford, 1976).  M1?????? ???

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