The amount of Tat protein present in the vial is shown on the label.

Tat protein stock:

Add suitable volume of Tris buffer (20 mM, pH 8) supplemented with 1 mM DTT to the vial and tap the vial to dissolve the protein at room temperature or use a plastic tip and pump the fluid up and down several times. It should not take more than a couple of minutes for the protein to go into solution. DTT prevents oxidation of Tat and must be used in the buffer. The original plastic vials were siliconised and should not permit protein loss by sticking to the surfaces. After ensuring that all the protein is gone into solution, stand the vials on ice. Use the protein as quickly as possible. Reconstitute Tat only on the day of your experiment.

Storing Tat solution:

You may store the protein solution for a week in the deep freezer (minus 80 degrees) but not beyond. Stored in a deep freezer, Tat retains nearly full biological activity (TNF-induction from primary monocytes) for a week. Nearly half the activity is retained up to one month (See Figure-8, Siddappa NB et al, 2006). Do not store Tat solution in minus-20-freezer or in the refrigerator.

Tat expression and purification:

The protein purification strategy consists of an affinity chromatography and an ion – exchange chromatography performed sequentially in that order. We amplified full length Tat from an Indian subtype – C clinical sample, cloned into an E. coli bacterial expression vector under the control of a T7 promoter and added a His – tag of 6 amino acid residues to the C-terminus of Tat to facilitate purification via Ni-NTA chromatography. E. coli cells expressing Tat were harvested by centrifugation and lysed by sonication. Bacterial lysate was subjected to two successive strategies of protein purification, Ni-NTA chromatography and Sp-Sepharose chromatography. Subtype-B Tat is derived from the molecular clone YU2.

Quality Control for HIV-1 subtype B and C Tat proteins:

1. SDS PAGE Analysis:

Tat protein concentration was determined by Bradford method and the NanoDrop spectrophotometer.
Figure-1: Tat proteins (5 and 10 µg) resolved on a 15% SDS-PAGE gel and stained with CBB.

2. Estimation of the endotoxin:

Endotoxin concentration in the recombinant Tat protein samples was determined using a commercial kit (QCL-1000, Biowhittaker). The concentration of endotoxin in these preparations was below the permitted limit (Table-1) of 0.10 EU/ µg (Ferreira GN et al, Trends Biotechnol, 18:380-388, 2000).

Table-1: Estimation of endotoxin

3. Endotoxin Estimation

Transactivation Assay:

HEK293 cells seeded in 12-well plates were transiently transfected with 0.5 µg of pLTR-GFP reporter vector using a standard calcium phosphate protocol. Twenty-four hours after the transfection, cells were incubated with freshly reconstituted Tat protein at a final concentration of 5 µg/ml in complete medium. Twenty-four hours following the protein transfection, expression of GFP was documented using UV-flourescence microscopy.

Figure-2: recombinant Tat-induced GFP expression from HIV-1 LTR.

4. Secretion of TNF-a from primary monocytes:

Monocytes were isolated from peripheral blood by differential density gradient centrifugation. The monocytes were seeded in 96-well plates at 1 × 104 cells/well. The culture medium was supplemented with 200 ng/ml of B-or C-Tat protein and incubated for 1 h. Cells were washed 3 times to remove Tat, resuspended in complete medium and incubated for 24 h. The level of TNF-a secreted into the culture medium was assessed by antigen capture ELISA using a commercial kit (R & D systems). As could be seen, both the Tat proteins induced secretion of Tat from the monocytes (Figure-3)

Some precautions:

Only use siliconised plastic tips and vials to dilute and handle Tat solutions. Tat is a sticky protein and sticks with high avidity to any surface. Protein losses will be significant if regular (non-siliconised) plastic materials are used.

Figure 3: Induction of cytokine secretion by Tat proteins. LPS (1 ng/ml) was used as a positive control for the cytokine induction.

Reference:

Please refer to the following publication for more information on Tat protein expression and functional standardization.

Siddappa NB et al, ‘Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein’, Retrovirology, 3, 53, 2006.

http://www.retrovirology.com/content/pdf/1742-4690-3-53.pdf

Protein batch made in: Dec 2007 Made by: Anand and Sundaram

Udaykumar Ranga
JNCASR, Bangalore, India
udaykumar@jncasr.ac.in