Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 92-93

REPORTE CORTO/SHORT REPORT

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

HIGH EXPRESSION LEVEL IN E. coli OF HTLV-I CORE PROTEINS

Marcelo Nazabal, Angel Perez and Ramon E. Narciandi.

Center for Genetic Engineering and Biotechnology. P.O. Box 6162, Havana 10600, Cuba.

Code Number: BA95033
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The Human T cell Leukemia Lymphoma virus type 1 (HTLV-I) was the first human retrovirus to be discovered (1). It is associated to adult T cell leukemia/lymphoma (ATLL) and a progressive neuromyelopathy named Tropical Spastic Paraparesis (TSP) (2). The antibodies of infected individuals are predominantly against the core proteins (p19 and p24) and the envelope protein gp46 (3). These proteins have been cloned in E. coli, but the expression levels obtained have been very low, less than 1% (4), in the specific case of p19-p24 and under the lactose promoter the expression level was 0.3% (5). In this work we cloned and expressed the core protein p19 and p24 fussed to IL-2 fragment, under tryptophan promoter, and the expression level obtained was 10% of total bacterial protein.

MATERIALS AND METHODS

The DNA fragment of 1042 bp corresponding to p19 and p24 proteins was amplified by PCR using specific primers. The fragment was cloned in the expression vector pR2M6 and transformed in the E. coli strain GC366. All the procedures for PCR, cloning and the analysis of the clones have been previously described (6). For the sequence analysis the Pharmacia sequencing Kit was used, following the manufacturer instructions. In one case, the induction of the tryptophan promoter was done in minimal media supplemented with glucose and casaminoacids at 37 C, by adding 0.012 mg/mL of beta-Indoleacrilyc acid after 2 h of the inoculation and growing the culture for 4 h at 37 C. Also, recombinant cells were inoculated in Luria Broth supplemented with 0.1 mg/mL of tryptophan and the culture was growth for 8 h at 37 C. The expression analysis was made by SDS-PAGE and Western blot (W.B) (7). In the latter, a sera from an infected patient diluted 1/10 in PBS-5% skim-milk and free of anti-E. coli antibodies, was used.

RESULTS AND DISCUSSION

The Plasmid named pGAG1, was obtained from the cloning of the PCR product corresponding to the p19-p24 gene in the pR2M6 expression vector. The clone was analyzed using the restriction enzymes XbaI and BamHI and by partial sequence of the ends of the gene, corroborating in frame insertion and absence of any mutation and stop codons. The induction of the clone in the E. coli strain GC366 by the two methods evaluated gave the following results. With minimal media and beta-Indoleacrilyc acid as inducer the expression was less than 1%, as previously reported for other cases. With rich media and the depletion of the repressor as induction method we obtained an increase in the expression level up to 10%. This result is better than those previously reported in E. coli and even in yeast, which was 3% (5). The HTLV-I positive sera used in the WB recognized only the protein at the expected molecular weight (4500 Da) in the lanes corresponding to the induced clones. These results allows the production of enough quantity of core antigens from HTLV-I, which is very important for the development of diagnostic kits for HTLV-I.

REFERENCES

1. B.. J. POIESZ et al.(1980) PNAS 77:7415.

2. G. de The and R. Bomford. (1993) AIDS Re. Hum. Ret. 65: 1870.

3. B. L. RENU et al. (1991) Journal of Virology 65: 1870.

4. S. ITARMURA et al. (1985) Gene 38:57.

5. Y. FUJISAWA et al.(1989) European Patent Application. 0. 352 060 A2.

6. J. SAMBROOK et al.(1989) Molecular Cloning: A. Laboratory Manual. Eds.Cold Spring Harbor Laboratories Press.

7. H. TOWBIN et al. (1989) PNAS 76: 4350.

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud