Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 90-91

REPORTE CORTO/SHORT REPORT

Presented in the Congress Biotecnologia Habana 94. La Habana, Cuba, Nov. 28 - Dec. 3, 1994

BACTERIAL EXPRESSION, ISOLATION AND PURIFICATION OF A DIAGNOSTIC RECOMBINANT PROTEIN REPRESENTATIVE OF HIV-1 gp120 (TAB1).

Ramon E. Narciandi, Abel Caballero, Denny Roque, Mayra Ponce, Vladimir Leal, and Lidia I. Novoa.

Center for Genetic Engineering and Biotechnology, Division of Immunotechnology and Diagnostics, P.O Box 6162, Cubanacan, Havana 10600, Cuba.

Code Number: BA95031
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The production of viral proteins by genetic engineering is by all means essential for the development of new diagnostic methods. The recombinant protein TAB1 is an artificial construction comprising an antigenic determinant common to HIV-1 gp120 plus several V3 loops from different viral isolates (1). Here we report a detailed process for the production and purification of this recombinant protein expressed as cytoplasmic insoluble inclusion bodies in E. coli.

EXPERIMENTAL PROCEDURES

The TAB1 insert was cloned into the pPF-15 vector (1), bearing a tryptophan promoter that drives expression of cloned genes as fusion proteins with 58 aa of human IL-2 in their N-terminus. The E. coli strain K-12 W-3110 was used as host. All fed-batch process was done in 50 L fermenters (B.E. Marubishi, Japan), at 37 C, 1 vvm, and 600 rpm. A complex media supplemented with 0.50 g/L of ampicillin was used. In the fed-batch step, the feed solution (yeast extract 12 g/L, glucose 200 g/L) was added when a cell weight of 0.37 g/L was achieved. In all cases the culture time was 12 h, and the induction of the tryptophan promoter was made by depletion of the initial tryptophan concentration of the culture medium. Cellular mass was reported as dry weight (g/L). Expression was estimated by SDS-PAGE densitometry. Cells were harvested by centrifugation. Cell pellets were suspended in TE buffer and lysed by sonication on ice. The isolated inclusion bodies were washed with different buffers and caotropic solutions (2). Finally, the inclusion bodies were completely solubilized in TE buffer containing 8 M guanidine and purified by RP-HPLC, using a C4 10x250 mm, 0.015 mm BAKERBOND (USA) reverse phase semipreparative column. For analysis, a C4 4.6 x 100 mm, 0.005 mm BAKERBOND (USA) reverse phase column was used. All purifications were done using linear elution gradients.

Fig. 1. Chromatography profile of TAB1 (gp120-r) by RP- HPLC. A- Initial soluble extract in a semipreparative column. B- Final purified material in an analytical column.

TAB1 is expressed in E. coli as insoluble inclusion bodies lacking a distinct membrane. After 8 hours of cultivation the expression of the recombinant protein started due to depletion of the initial tryptophan concentration. The highest yield was obtained after 12 h of culture. At this time, 10 g of cells/L were obtained, with TAB1 being 20% of the total cellular protein. The intact granules separated from the cellular lysates by centrifugation were semipurified by a wash pellet cell procedure. Washing the pellet with different solutions removes a great quantity of insoluble impurities and some of the cellular debris. Using this procedure a material with a purity higher than 70% was obtained. The purification of the TAB1 was done by reverse-phase HPLC. The larger collected fraction in figure 1A corresponds with TAB1, and is easily separated from other proteins. The purified antigen (figure 1B; 97% purity) has been used in the development of a diagnostic assay system, showing good specificity and sensitivity.

REFERENCES

1. DUARTE, C. et al. (1994). AIDS Res. Hum. Retrov. 10, (3): 235-243.

2. NARCIANDI, R. E. et al. (1993). Biotecnologia Aplicada 10 (1): 36-40.

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud

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