Disulphide bridge formation in the periplasm of Escherichia coli: beta-lactamase:: human IgG3 hinge fusions as a model system.
We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla). In the first fusion gene blahi, TEM1 beta-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer. The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.
De Sutter
K
K
Laboratory of Molecular Biology, Gent University, Belgium.
Remaut
E
E
Fiers
W
W
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D017385:Sequence Homology; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004220:Disulfides; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases