Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 91-92

REPORTE CORTO/SHORT REPORT

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

A RECOMBINANT TMPA FUSION PROTEIN. EFFECT OF IL-2 AND HISTIDIN DOMAINS ON EXPRESSION

Racmar Casalvilla, Maria del C. Dominguez, Marta Duenas.

Center for Genetic Engineering and Biotechnology. P.O. Box 6162, Havana 6, Cuba.

Code Number: BA95032
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Treponema Membrane Protein A (TMPA) is one of the most successful diagnostic markers for Syphilis (1). A recombinant TMPA has been expressed by us in E. coli as a cytoplasmic soluble protein, using the pPF-15 vector (2). The resulting protein is a fusion of the 345 aa of TMPA and 58 aa of human IL-2 at its N- terminus (3). Its nature and characteristics have made difficult to scale-up the purification process. In this report we explore the addition of histidine domains to the protein, as a way to use immobilized Metal Affinity Chromatography for purification. We also evaluate the importance of the IL-2 fragment in expression.

MATERIALS AND METHODS

Plasmid construction

The pPF-15 plasmid (2), bearing the tryptophan promoter, the T4 terminator, and the N-terminus IL-2 fragment was modified so as to: (a) include 6-histidine domain encoding sequences either at the amino- or carboxi terminus of the expressed protein, (b) remove the IL-2 sequence, while including the aforementioned histidine domains,. The TMPA encoding DNA sequence, obtained by PCR, was cloned into the five different plasmids, and used to transform the E. coli strain W3110. The cells were plated in 2XYT medium with 0.1 mg/mL of ampicillin, and colonies screened by PCR. Selected positive clones were checked by sequence to determine correct frame for the amino and carboxiterminal histidine domains.

Expression

Positive clones were grown in 10 mL 2XYT medium with 0.1 mg/mL of ampicillin for 8 h, followed by the inoculation at 0.1 O.D. of 50 mL of M9 medium, supplemented with 0.5% of glucose and 0.5% of casaminoacids. Four hours later, expression was induced by adding 0.02 mg/mL of 3-b indoleacrylic acid and the culture was allowed to grow for four more hours. Expression was checked by using SDS-PAGE and Western blot (W.B.) using a reactive sera pool obtained from ten infected patients.

RESULTS AND DISCUSSION

All constructions gave positive clones, as shown by PCR and sequence. In every case, at least 5 clones were used to test expression levels. No expression could be demonstrated by SDS- PAGE or W.B. when the N-terminus IL-2 fragment encoding sequence was absent. If the IL-2 sequence was present, but preceded by a six-histidine domain, expression was very low. Only the construction with the IL-2 fragment N-terminus sequence, an the six histidine domain at the carboxiterminus showed high expression levels of TMPA (20% of total bacteria protein), and the protein was recognized by the specific serum in W.B.

We, and others, have discussed that the presence of a human IL-2 encoding sequence, 5' to the protein of interest, provides an excellent stabilizer for protein expression in E. coli, either by conferring high stability to mRNA, or by reducing the probability of proteolysis (2, 4-5). This same effect has been found with the five other different viral proteins, and immunoglobulin fragments we have cloned into pPF-15, and its modified versions lacking the IL-2 fragment (unpublished results). Now we also show that the effect of the IL-2 sequence at the N-terminus can be partially affected by a preceding six- histidine domain.

REFERENCES

1. YOUNG, H. et al (1992). International Journal of SDT and AIDS 3:391-413

2. EUROPEAN PATENT APPLICATION N.90202108.8

3. DOM NGUEZ, M. C. et al. (1994) Advances in Modern Biotechnology 2:93. Biotecnologia Habana'94. Short Reports of the Congress, La Habana, Cuba, Nov.28-Dic.3,1994

4. DEUTSCHER, M. P. et al (1988). TIBS 13:103-115

5. LAVALLIE, E. R. et al. Biotechnology 11: 187-192

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud