Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 2, 1995, pp. 97-98

REPORTE CORTO/SHORT REPORT

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

VARIABLE REGION SEQUENCES MODULATES PERIPLASMIC EXPORT OF A scFv ANTIBODY FRAMENT, SPECIFIC FOR HEPATITIS-B SURFACE ANTIGEN, IN E. coli

Marta Ayala1, Maria E. Fernandez-de-Cossio1, Leonardo Cannaan- Haden1, Robert F. Balini2, James W. Larrick2, Jorge V. Gavilondo1.

1Center for Genetic Engineering and Biotechn, P.O.Box 6162, La Habana, Cuba. 2Palo Alto, Institute of Molecular Medicine, USA

Code Number: BA95038
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Recent developments in recombinant DNA technology make possible the production of Fab, Fv, and single-chain Fv (scFv) antibody fragments in genetically engineered microorganisms (1). Applications of recombinant Fab and scFv include the elucidation of antigen-antibody interaction, imaging, drug and toxin targeting, catalysis, immunomodulation, immunotherapy, neutralization, and detoxification. Recently authors have also show that antibody fragments could have potential for the immunopurification (2). We have produced a bacterial scFv specific for a recombinant Hepatitis B virus surface antigen (HBsAg). Using expression vectors designed for periplasmic export we found that the scFv nevertheless remained associated to bacterial insoluble material. We will show evidence suggesting that positively charged aminoacids of the heavy chain V-region (VH) could be responsible for the association of the scFv to the bacterial inner membrane.

EXPERIMENTAL PROCEDURES

RNA was extracted from the anti-HBsAg mouse hybridoma CB-Hep.1, and cDNA synthesized. PCR was used for the assembly of the scFv (VL-L-VH or VH-L-VL) gene, including a 14 aminoacid spacer between VH and VL regions, and for site-directed mutagenesis of the VH domain. The sequenced gene was inserted for export into the secretion vectors pPACIB.1 and pPACIB.3, bearing OmpA secretion signal and for intracellular expression into pPACIB.4 and pPACIB.5 vectors. All vectors bear 6-histidine coding domain that are fused at the N- or C- terminal of the mature protein. Several E. coli strains were transformed and expression induced with beta indoleacrylic acid. Western Blots (WB) of SDS- PAGE bacterial material were developed with a specific anti Fab rabbit antibody. The activity of fragments was monitored by specific ELISA against HBsAg. The proteins were purified by immobilized metal affinity chromatography (IMAC).

RESULTS AND DISCUSSION

A high expression level was found for the scFv (VH-L-VL) in pPACIB.1 and pPACIB.3 when strain MM294 was used (ca. 15% of total bacterial protein). While both vectors have a bacterial secretion signal, the synthesized protein is not secreted into the periplasm. Extraction studies and electron microscopy indicate that the scFv is associated to the insoluble membrane fraction. This fraction was solubilized with 6M Urea, and the scFv with a 60% of purity renatured by extensive dyalisis against PBS. The renatured scFv binds to the antigen in a direct ELISA. Two different versions of the scFv fragment were cloned into the pPACIB.1 vector; one of them with mutated VH domain (Arg 16-Gly) and the other with change in domain order (VL-L-VH). These changes brought forth the export active scFv to periplasm, suggesting that framework 1 positively charged aminoacids of the VH region could be responsible for the association of the scFv to the bacterial membrane, as have been suggesting by other different studies (3). A succesful intracellular expression was confirmed when the pPACIB.5 vector was used, while no expression was detected when the pPACIB.4 vector was employed. These results indicate that the human IL-2 coding region (58 aa) 5' to the protein, is a potent factor in succesful expression of heterologous protein in bacteria.

REFERENCES

1. PLUCKTUN, A. et al. (1991). Biotechnology 9:273-278

2. PIERCE, J. .J. et al. (1993). J. of Chrom. 629:161-168

3. BECKWITH, J. and D. BOYD (1990). Cell 62:1031- 1033

Copyright 1995 Sociedad Iberolatinoamericana de Biotecnologia Aplicada a la Salud