Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis
| Autor: | J.B. Molfetta, Anderson Miyoshi, E.D.C. Gonçalves, Vasco Azevedo, Cristina Silva, Clarissa Santos Rocha, N. Electo, M.S.P. de Azevedo, Daniela Santos Pontes |
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| Rok vydání: | 2012 |
| Předmět: |
Heterologous
Polymerase Chain Reaction Microbiology law.invention Immune system Bacterial Proteins law Heat shock protein Genetics Extracellular Cloning Molecular Molecular Biology Mycobacterium leprae DNA Primers biology Base Sequence Chemistry Lactococcus lactis General Medicine Chaperonin 60 biology.organism_classification Recombinant Proteins Limulus amebocyte lysate Recombinant DNA |
| Zdroj: | Genetics and molecular research : GMR. 11(2) |
| ISSN: | 1676-5680 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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