Production of lipidated meningococcal transferrin binding protein 2 in Escherichia coli.
Neisseria meningitidis strains grown under iron starvation conditions produce transferrin binding proteins (Tbp1 and Tbp2) which have been shown to play a major role in iron acquisition. Recent studies performed with Tbp2 purified from N. meningitidis suggest that this surface protein is a potential vaccine component. In order to further evaluate the immunogenicity of Tbp2, it was essential to develop a heterologous expression system to generate high amounts of purified protein. Tbp2 is produced in Neisseria as a precursor with a signal peptide whose cleavage follows a lipidation step on a cysteine residue which is the first amino acid in the mature protein. When produced in Escherichia coli with its natural signal peptide, a high amount of Tbp2 (about 10% of total cell proteins) was detected. However, most of the protein was nonlipidated precursor and only a small fraction was mature Tbp2. In order to optimize the maturation of the precursor, the natural signal sequence was replaced by several E. coli lipoprotein signal peptides. Expression levels and maturation of the precursor were highly variable depending on the signal peptide used. With one of these, an efficient maturation and a high amount of mature lipidated Tbp2 were obtained (about 3% of total cell proteins). A large-scale production process was then established for this E. coli-produced Tbp2.
Legrain
M
M
Transgene SA, Strasbourg, France.
Speck
D
D
Jacobs
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics*; D033862:Iron-Binding Proteins; D008074:Lipoproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000737:chemistry*; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D014168:Transferrin / Q000378:metabolism; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins
D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D008074:Lipoproteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D014168:Transferrin; D033882:Transferrin-Binding Protein B; D033863:Transferrin-Binding Proteins; D003545:Cysteine