       Document 0171
 DOCN  M9610171
 TI    Studies of the anaerobically induced promoter pnirB and the improved
       expression of bacterial antigens.
 DT    9601
 AU    Newton SM; Klebba PE; Hofnung M; Charbit A; Departemento de
       Microbiologia, Universidade de Sao Paulo,; Brazil.
 SO    Res Microbiol. 1995 Mar-Apr;146(3):193-202. Unique Identifier : AIDSLINE
       MED/96008858
 AB    The promoter of the Escherichia coli gene nirB is induced by both the
       presence of nitrite in the environment and by low oxygen tensions. It
       has been used to direct the high-level expression of heterologous
       proteins by E. coli strains in fermentors, and attenuated Salmonella
       strains expressing foreign proteins under nirB promoter (pnir) control
       have efficiently induced an immune response against these proteins. The
       genes encoding two different E. coli envelope proteins, the outer
       membrane protein LamB and the periplasmic protein MalE, were placed
       under pnir control on pBR322 derivatives, and both proteins were
       expressed at high levels during anaerobic growth. Our results showed
       that the expression level of MalE was influenced by the distance between
       the pnir promoter and the Shine-Dalgarno sequence: the highest levels
       were obtained by the longest constructs made; pnir directed a 4-fold
       increase in the level of MalE expression relative to the level reached
       by the previously described ptac-MalE expression vector. The best pnir
       construct produced 25 mg of MalE protein per 5 x 10(11) bacteria, which
       represents over 20% of total cell protein. Overexpression of MalE was
       well tolerated by E. coli, even under strict anaerobic conditions; for
       LamB, optimal induction was achieved under partial anaerobiosis. A
       MalE-HIV1 hybrid protein (33 residues from the V3 loop of HIV1 gp160
       inserted into site 133 of MalE) was also overexpressed at a similar
       yield under pnir control, without apparent degradation of the hybrid
       protein. Moreover, when expressed in attenuated aroA S. typhimurium
       strain SL3261, the plasmids carrying malE and malE-HIV genes were stable
       in vitro and in vivo.
 DE    Anaerobiosis  Antigens, Bacterial/*GENETICS  Bacterial Outer Membrane
       Proteins/ANALYSIS/GENETICS  Bacterial Proteins/ANALYSIS/GENETICS
       Electrophoresis, Polyacrylamide Gel  Escherichia
       coli/*GENETICS/IMMUNOLOGY  Gene Expression Regulation, Bacterial  Genes,
       Bacterial/*GENETICS  In Vitro  Plasmids  Promoter Regions
       (Genetics)/*GENETICS  Salmonella typhimurium/*GENETICS/IMMUNOLOGY
       Support, Non-U.S. Gov't  Support, U.S. Gov't, Non-P.H.S.  JOURNAL
       ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

