Targeting of interleukin-2 to the periplasm of Escherichia coli.
A synthetic gene coding for interleukin-2 (IL-2) was used to produce large amounts of recombinant IL-2 (met-IL-2) in Escherichia coli. Met-IL-2 was found to accumulate in the cytoplasm in an insoluble, aggregated form. Inclusion bodies located at the pole caps of cells were detected using immunogold labelling. Constructs were designed to fuse the IL-2 gene to DNA fragments encoding signal peptides for an outer-membrane protein (OmpA) or for a periplasmic protein (PhoA) of E. coli. No significant maturation was observed with these fusion proteins which were found in an insoluble form in the cytoplasm. The influence of charge disposition at the N-terminus of the mature portion of the protein was investigated by replacing positively charged amino acids with glutamic acid. None of the introduced substitutions had any effect. Various factors that might affect expression, secretion and folding were examined in an attempt to obtain secretion. By fusing IL-2 to the precursor maltose-binding protein (preMBP) a large fraction of the preMBP-IL-2 protein was correctly processed and transported to the periplasmic space. IL-2 derived from MBP-IL-2 after FXa cleavage possessed similar specific activity to recombinant IL-2 produced in Chinese Hamster ovary cells.
Halfmann
G
G
Laboratoire d'Ingénierie et Dynamique des Systèmes Membranaires du CNRS, Marseille, France.
Brailly
H
H
Bernadac
A
A
Montero-Julian
F A
FA
Lazdunski
C
C
Baty
D
D
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D007376:Interleukin-2; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins