Brazilian Journal of Medical and Biological Research Volume 38

Brazilian Journal of Medical and Biological Research
Volume 38, Issue 11, November 2005, Pages 1585-1592

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

Freitas, D.A., Leclerc, S., Miyoshi, A., Oliveira, S.C., Sommer, P.S.M., Rodrigues, L., Correa Jr., A., Gautier, M. Langella, P., Azevedo, V.A., Le Loir, Y.

^aLaboratory of Microbiology, UMR1253 STLO, Agrocampus INRA, 65 rue de Saint Brieuc, 35042 Rennes Cedex, France
^bInstituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
^cDepartamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
^dLaboratory of Applied Genetics, Unit of Dairy Research and Applied Genetics, National Institute for Agricultural Research, Domaine de Vilvert, Jouy en Josas Cedex, France

Abstract

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae and fungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Author Keywords

Antifungal protein; Lactococcus lactis; Secretion; Streptomyces tendae antifungal protein 1

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