title: Cytoplasmic and extracellular expression of  pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus  lactis.
abstract: Lactic acid bacteria (LAB) are an attractive and safe  alternative for the expression of heterologous proteins, as they are  nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB  model organism, has been extensively employed in the biotechnology field for  large-scale production of heterologous proteins, and its use as a "cell  factory" has been widely studied. We have been particularly interested in the  use of L. lactis for production of heat shock proteins (HSPs), which  reportedly play important roles in the initiation of innate and adaptive immune  responses. However, this activity has been questioned, as LPS contamination  appears to be responsible for most, if not all, immunostimulatory activity of  HSPs. In order to study the effect of pure HSPs on the immune system, we  constructed recombinant L. lactis strains able to produce and properly  address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or  to the extracellular medium, using a xylose-induced expression system. Approximately  7 mg/L recombinant Hsp65 was secreted. Degradation products related to  lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate  assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations  was 10-100 times lower than the permitted levels established by the U.S. Food  and Drug Administration. These new L. lactis strains will allow  investigation of the effects of M. leprae Hsp65 without the interference  of LPS; consequently, they have potential for a variety of biotechnological,  medical and therapeutic applications.
authors.affiliation: Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
authors.forename: M S P
authors.initials: MS
authors.lastname: de Azevedo
authors.forename: C S
authors.initials: CS
authors.lastname: Rocha
authors.forename: N
authors.initials: N
authors.lastname: Electo
authors.forename: D S
authors.initials: DS
authors.lastname: Pontes
authors.forename: J B
authors.initials: JB
authors.lastname: Molfetta
authors.forename: E D C
authors.initials: ED
authors.lastname: Gonçalves
authors.forename: V
authors.initials: V
authors.lastname: Azevedo
authors.forename: C L
authors.initials: CL
authors.lastname: Silva
authors.forename: A
authors.initials: A
authors.lastname: Miyoshi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D009166:Mycobacterium leprae / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; D011994:Recombinant Proteins; C074119:heat-shock protein 65, Mycobacterium