Expression of an 18 kDa::PhoA fusion protein in Mycobacterium spp

Christopher W. Robb, Haolin Ni, Heiman Wang, Alan Barrett, David Niesel

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Recent advances with mycobacterial vectors hold promise for the development of recombinant mycobacterial vaccines. Production of heterologous proteins by mycobacteria can elicit strong cellular and humoral immune responses. Importantly, expression of proteins at the surface of Mycobacterium spp. results in significant humoral responses as compared to those against cytoplasmic proteins. We have developed pCR7, a plasmid vector that expresses the M. leprae 18 kDa antigen fused in-frame to E. coli alkaline phosphatase (PhoA). The fusion sequence is flanked by insertion sequence (IS900) elements, allowing stable integration into the mycobacterial chromosome. A 59-kDa protein, the predicted size of the fusion product, was detectable by immunoblotting with monoclonal antibody to PhoA and to the 18 kDa antigen. M. smegmatis and M. vaccae transformed with pCR7 exhibited alkaline phosphatase (PhoA) activity, indicating transport of the heterologous protein across the mycobacterial membrane. pCR7 transformants: (a) had a single copy of the gene construct, (b) varied in the level of PhoA activity and (c) were cultivated stably in the absence of antibiotic pressure. Furthermore, production of the 18 kDa::PhoA fusion protein in pCR7 transformants was significantly enhanced during intracellular incubation in J774 macrophage monolayers. Thus, pCR7 may offer several advantages as a recombinant vaccine vector. Target antigens can be expressed in-frame with the 18 kDa::PhoA construct. Such recombinant Mycobacterium spp. would express the target antigen at the mycobacterial surface, co-express the immunostimulatory M. leprae 18 kDa sequences, and allow enhanced production of target antigens in vivo. Importantly, production of heterologous proteins could be verified by screening for PhoA activity, providing a potential alternative to antibiotic selection. Copyright (C) 1998 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)245-254
Number of pages10
JournalJournal of Microbiological Methods
Volume33
Issue number3
DOIs
StatePublished - Aug 1 1998

Fingerprint

Phosphoprotein Phosphatases
Mycobacterium
Phosphoric Monoester Hydrolases
Antigens
Synthetic Vaccines
Alkaline Phosphatase
Proteins
Smegma
Anti-Bacterial Agents
DNA Transposable Elements
Humoral Immunity
Immunoblotting
Cellular Immunity
Carrier Proteins
Membrane Proteins
Plasmids
Chromosomes
Macrophages
Monoclonal Antibodies
Escherichia coli

Keywords

  • 18 kDa
  • Alkaline phosphatase
  • Heterologous protein
  • Mycobacteria
  • Recombinant DNA

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

Expression of an 18 kDa::PhoA fusion protein in Mycobacterium spp. / Robb, Christopher W.; Ni, Haolin; Wang, Heiman; Barrett, Alan; Niesel, David.

In: Journal of Microbiological Methods, Vol. 33, No. 3, 01.08.1998, p. 245-254.

Research output: Contribution to journalArticle

Robb, Christopher W. ; Ni, Haolin ; Wang, Heiman ; Barrett, Alan ; Niesel, David. / Expression of an 18 kDa::PhoA fusion protein in Mycobacterium spp. In: Journal of Microbiological Methods. 1998 ; Vol. 33, No. 3. pp. 245-254.
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