Chaperone DnaJ is a homodimer with each subunit containing 10 cysteine residues and two Zn(II) ions, which have been identified to form two zinc fingers, C(144)DVC(147)Zn(II)C(197)NKC(200) (Zn1) and C(161)PTC(164)Zn(II)C(183)PHC(186) (Zn2), with C(265) and C(323) in reduced form. Guanidine hydrochloride at 6.4 M destroys only Zn1, which does not reform after refolding. p-Hydroxymercuriphenylsulfonate acid, but not ethylenediaminetetraacetic acid (EDTA) even at high concentrations, can remove two Zn(II) ions from DnaJ, but only Zn2 can be reconstituted. After removal of Zn(II) ions, only C(144) and C(147) in Zn1 are oxidation-resistant, and the other six cysteines are easily oxidizable. DnaJ shows reductase activity and oxidase activity but little, if any, isomerase activity. The reductase activity is reversibly inhibited by EDTA. Zn2 is important for the enzymatic activity, and only -C(183)PHC(186)- among the four motifs of -CXXC- functions as the active site of the enzyme. A C-terminal (Q(181)-R(376)) fragment shows a zinc finger of C(183)PHC(186)Zn(II)C(197)NKC(200) and full enzymatic activity of DnaJ. The N-terminal half sequence (M(1)-Q(180)) and Zn1 are not required for the enzymatic activity but are important for the chaperone activity of DnaJ.

Escherichia coli DnaJ, possessing both chaperone and thiol-disulfide oxidoreductase activities, is a homodimeric Hsp40 protein. Each subunit contains four copies of a sequence of -CXXCXGXG-, which coordinate with two Zn(II) ions to form an unusual topology of two C4-type zinc fingers, C144DVC147Zn(II)C197NKC200 (Zn1) and C161PTC164Zn(II)C183PHC186 (Zn2). Studies on five DnaJ mutants with Cys in Zn2 replaced by His or Ser (C183H, C186H, C161H/C183H, C164H/183H, and C161S/C164S) reveal that substitutions of one or two Cys residues by His or Ser have little effect on the general conformation and association property of the molecule. Replacement of two Cys residues by His does not interfere with the zinc coordination. However, replacement of two Cys by Ser results in a significant decrease in the proportion of coordinated Zn(II), although the unique zinc finger topology is retained. The mutants of C183H, C186H, and C161S/C164S display full disulfide reductase activity of wild-type DnaJ, while C161H/C183H and C164H/183H exhibit severe defect in the activity. All of the mutations do not substantially affect the chaperone activity. The results indicate that the motif of -CXXC- is critical to form an active site and indispensable to the thiol-disulfide oxidoreductase activity of DnaJ. Each -CXXC- motif in Zn2 but not in Zn1 functions as an active site.

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis-trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis-trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.

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