ISSN:
1365-2958
Quelle:
Blackwell Publishing Journal Backfiles 1879-2005
Thema:
Biologie
,
Medizin
Notizen:
We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human lgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 β-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3′ end of the β-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° C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL 1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that β-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 β-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-nke 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.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1111/j.1365-2958.1992.tb01394.x
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