title: Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by Lactococcus lactis.
abstract: BACKGROUND
  Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10.
  
  
  RESULTS
  The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes.
  
  
  CONCLUSIONS
  Expression and secretion of mature IP-10 was efficiently achieved by L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant L. lactis represents a potentially useful tool to be used as a live vaccine in vivo.
authors.affiliation: Departamento de Histología, Facultad de Medicina, Universidad Autónoma de Nuevo León (UANL), Monterrey, N,L,, México. rrrmontes@yahoo.com.
authors.forename: Julio
authors.initials: J
authors.lastname: Villatoro-Hernandez
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Loera-Arias
authors.forename: Anali
authors.initials: A
authors.lastname: Gamez-Escobedo
authors.forename: Moises
authors.initials: M
authors.lastname: Franco-Molina
authors.forename: Jorge G
authors.initials: JG
authors.lastname: Gomez-Gutierrez
authors.forename: Humberto
authors.initials: H
authors.lastname: Rodriguez-Rocha
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Gutierrez-Puente
authors.forename: Odila
authors.initials: O
authors.lastname: Saucedo-Cardenas
authors.forename: Jesus
authors.initials: J
authors.lastname: Valdes-Flores
authors.forename: Roberto
authors.initials: R
authors.lastname: Montes-de-Oca-Luna