Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Roger A. Laine

Abstract

The nucleotide sequence of the gene encoding the cytoplasmic $N,N\sp\prime$-diacetylchitobiase from Vibrio parahaemolyticus has been determined. The deduced peptide sequence surprisingly has minimum evolutionary relationship to two other reported $N,N\sp\prime$-diacetylchitobiases from vibrios, except for highly conserved regions which are also homologous with lysosomal beta-hexosaminidases from eukaryotes including humans. In contrast, the other two sequenced chitobiases from vibrios are much more closely related to each other. This 85 kDa cytoplasmic protein, as revealed from the sequence, appears to be a unique protein, lacking a signal sequence and genetically distant from other known enzymes of similar function. This is consistent with its limited substrate specificity to small N-acetylglucosamine terminated oligosaccharides. The signal peptide of an extracellular endochitinase from V. parahaemolyticus causes the mature chitinase to be efficiently secreted through the double membranes of both Gram negative V. parahaemolyticus, and of Escherichia coli JM101 when the gene is cloned therein. By using recombinant PCR, this signal sequence was fused in frame to the chitobiase coding sequence, and active chitobiase was found in the E. coli culture medium. Two secretion vectors were developed during this study for the secretion of wild type proteins. PCR fragment of a structural gene can be inserted in frame with the signal sequence, which is under the strong trc promoter. The secretion system has been tested using the cytoplasmic chitobiase gene as a model system. Restriction sites and complete DNA sequence are not required for the structural gene to be cloned. The chitobiase and the original chitinase as cloned for secretion were stabilized by EDTA added to the medium. This observation may prove generally useful for protecting cloned and secreted proteins in E. coli.

Pages

140

Share

COinS