Construction of Candida
albicans Two-hybrid Library and Screening for Proteins Interacting with
Crk1
NI Jian, CHEN Xi, YANG Tao, CHEN Jiang-Ye*
( State Key Laboratory of Molecular Biology, Institute of Biochemistry and
Cell Biology, Shanghai Institute for Biological Sciences, the Chinese Academy
of Sciences, Shanghai 200031, China )
Abstract In order to
study interactions among proteins involved in Candida albicans
morphorgenesis, C.albicans genomic DNA was digested with Sau3AI
and fused to activating domain(AD) of LEXA to construct an AD-fused C.albicans
genomic DNA expression library. The library contained 7.7×104
independent clones and included 1.2×105 kb DNA which was 10 fold of
the C.albicans whole genomic DNA. The CRK1 gene encodes a
CDC2-related protein, that was involved in morphorgenesis of C.albicans.
The Crk1 fused to DNA binding domain of LexA was used as the bait to screen the
LexA library. Among 9×105 transformants, eight Leu and LacZ
double positive clones were selected. With restriction enzyme analysis, three
kinds of fragments were identified. The clones NJ1, NJ2, NJ3 were
sequenced and analyzed with BLAST program. It was found that NJ1,NJ2,
NJ3 were homologs of S.cerevisiae gene STI1,RET2
and NFI1, respectively. The kinase domain of Crk1(Crk1N)interacted
weakly with protein encoded by NJ1, while the noncatalytic domain of
Crk1(Crk1C) interacted strongly with proteins encoded by NJ2 and NJ3.
The interaction of NJ3 and Crk1C was verified by the coimmunoprecipition
of NJ3-HA with Crk1C-LexA. These proteins may be involved in maturation,
transport, localization, and activity regulation of the Crk1.
Key words Candida albicans； CRK1； LexA two-hybrid system
*Corresponding author： Tel, 86-21-64374430； Fax, 86-21-64338357； e-mail, [email protected]