Cloning and functional expression of ubiquitin-like protein specific proteases genes from Candida albicans.
The small ubiquitin-like modifier (SUMO) modification occurred at bud necks and sites of septum formation in hyphae of the opportunistic fungal pathogen Candida albicans. Three genes encoding putative SUMO deconjugation enzymes (Ulp, ubiquitin-like protein specific proteases) of C. albicans were obtained through sequence database searching with Ulp domain of Saccharomyces cerevisiae Ulp1 (ScUlp1). These genes were designated as CaULP1, CaULP2 and CaULP3. The open reading frames of three putative ULPs were cloned and expressed in Pichia pastoris, resulting recombinant proteins. Functional analysis of recombinant CaUlp1, CaUlp2 and CaUlp3 confirms that these proteins exhibit SUMO-processing activity. CaULP1, CaULP2 and CaULP3 only expressed active form enzyme in P. pastoris but not in Escherichia coli. The molecular weights of CaUlp1, CaUlp2 and CaUlp3 proteins expressed in P. pastoris were larger than theoretical molecular weights. This observation was in good agreement with result of Western blot analysis of CaUlp1 and CaUlp3 proteins in C. albicans. It was assumed that CaUlp1, CaUlp2 and CaUlp3 proteins may need post-translational modifications to exhibit SUMO-processing activity. To our knowledge, this is the first report on cloning and expression of Ulp genes from C. albicans. Furthermore RT-PCR and Western blot analysis show that CaULP2 has no detectable expression both in yeast and in hyphal forms of C. albicans.
Huaping
Li
L
School of Life Science, Sun Yatsen University, Guangzhou, P.R. China. li.huaping@gmail.com
Jie
Lian
L
Zhifeng
Wang
W
Yingchang
Zhong
Z
Yuhuan
Liu
L
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002176:Candida albicans / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D025301:Hyphae / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000096:biosynthesis / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins