The expression of murine protein disulfide isomerase in Escherichia coli.
Protein disulfide isomerase (PDI), a luminal enzyme of the endoplasmic reticulum (ER), is thought to be involved in the process that assures that the correct disulfide bonds form as a newly synthesized protein folds into its appropriate three-dimensional structure (Freeman, 1984). In recent years, the ER has been shown to have at least two additional, distinct PDI-related luminal proteins (Bennett et al., 1988; Mazzarella et al., 1990). As a potential first step toward an investigation of the structure and function of PDI and of the PDI-related proteins as well, we have developed a bacterial expression system in Escherichia coli capable of synthesizing significant levels of enzymatically active PDI under the control of the inducible tac promoter. We have observed that the use of this bacterial expression system is complicated by the fact that there is a significant amount of internal initiation of protein synthesis within the PDI coding sequence and the fact that all of the PDI-related expression products are found equally distributed between the cytoplasmic and periplasmic fractions due to a single peptide-independent mechanism. Our studies with this system have demonstrated that at least some truncated PDI molecules containing the carboxy-terminal most active site have significant PDI activity.
Haugejorden
S M
SM
Department of Microbiology, Saint Louis University Medical Center, MO 63104.
Srinivasan
M
M
Green
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007535:Isomerases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases