| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 87 === The Sec system is the major pathway required for the secretion of proteins in many bacteria. It has been showed that the core enzyme of Sec system is composed of SecY, SecE and SecG. The goal of this study is to clone and characterize the secE and secY from Xanthomonas campestris pv. campestris (Xc11). The nucleotide sequence of the previously isolated pBKBet-1 plasmid that containing the upstream region of the Xc11 rpoB was determined. Results of the DNA sequence analysis revealed that the genomic location of the secE may be immediately upstream of the rpoB operon and have the same gene order as that in E. coli. By using the EcoRI-XhoI 0.7 kb fragment of pBKBet-1 that containing nusG and part of the secE as probe, a recombinant l phage that bearing the intact secE gene was isolated from the constructed Xc11 genomic library. In vivo excision was performed to generate pBK-CMV derivated phage from the recombinant l phage and designated as pBKSec14. The nucleotide sequences on both strands of the 1.2-kb insert were determined. Results of the DNA sequence analysis revealed two open reading frames (ORF). The first ORF (ORF136) begins at nt 296 and ends at nt 686, from which a 136 amino acids with molecular weight of 15.2 kDa could be translated. A GTG codon at nt 329 was suggested as the start codon of the same ORF by primer extension analysis. This ORF (ORF118) encodes a 118 amino acids protein with a molecular weight of 13.4 kDa. Both of the ORF 136 and ORF118 showed 48.7% amino acid sequence identities with that of the E. coli. The protein encoded by ORF118 has been predicated to have two transmembrane domains (TMs) and has a signal peptidase cleavage site at the 21st amino acid. The second ORF (ORF186) begains at nt 700 and ends at nt 1257 from which a 186 amino acids could be encoded. To overexpress SecE protein, the secE gene was PCR amplified and subsequently cloned into pET21bT vector. The SecE with six histidine amino acids at its C-terminus was purified through His-bind resin. The purified SecE would be used as an antigen to produce antiserum for Western blot analysis. Moreover, the DNA sequence immediatedly upstream of the S13 gene of the Xc11 rpoA operon was also determined. Results of the 3158-bp DNA sequence analysis revealed that there are six ORFs (L30-L15-SecY-S13-S11-S4) and have the same gene order as that of the E. coli. The ORF455 begins at nt 578 and ends at nt 1945, from which a 455 amino acid with molecular weight of 48.4 kDa could be translated. The Xc11 SecY showed 56.3% identity with the E. coli SecY protein. Unfortunately, the protein encoded by the full length DNA fragment of secY gene can not be overexpressed in E. coli.
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