[Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system].
OBJECTIVE
To investigate the interaction domains between macrophage migration inhibitory factors (MIF) and the extracellular segment of type-II trans-membrane protein CD74 using a yeast two-hybrid system.


METHODS
By using molecular cloning techniques, the DNA fragments encoding MIF, MIF(50-65) and MIF(1-50/65-115) were introduced into the pGBKT7 vector to construct the corresponding recombinant bait plasmids, and the DNA fragments encoding CD74(73-232), CD74(73-109), CD74(1109-149) and CD74(149-232) into the pGADT7 vector to construct the recombinant activation domain (AD) plasmids. PEG/LiAC method was employed to transform the above 3 recombinant bait plasmids paired with each of the 4 recombinant AD plasmids into the chemical competent yeast AH109 cells. The transformed yeast AH109 cells were screened consecutively on SD/-Trp-Leu and SD/-Trp-Leu-Ade-His/X-alpha-gal nutritional media.


RESULTS
The results of restriction endonuclease digestion and DNA sequencing verified the correct construction of all the recombinant plasmids. The yeast AH109 cells transformed with each of the 3 recombinant bait plasmids could grow on SD/-trp nutritional media without autonomous activation effect on the reporter gene MEL1. The cells transformed with each of the 4 recombinant AD plasmids could also grow on SD/-leu nutritional media without activation of the reporter gene MEL1. Only the yeast AH109 cells co-transformed with MIF, MIF(50-65), or MIF(1-50/65-115) plasmid and CD74(73-232) plasmid could grow on SD/-Trp-Leu-Ade-His nutritional media with transcription activation of the reporter gene MEL1.


CONCLUSIONS
MIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).
Shan
Zhi-xi
ZX
Research Center of Medical Sciences, Guangdong Provincial People's Hospital, Guangzhou/Guangdong Provincial Cardiovascular Disease Institute, Guangzhou 510080, China. zhixinshan@yahoo.com.cn
Lin
Qiu-xiong
QX
Deng
Chun-yu
CY
Tan
Hong-hong
HH
Kuang
Su-juan
SJ
Xiao
Ding-zhang
DZ
Zhu
Jie-ning
JN
Fu
Yong-heng
YH
Yu
Xi-yong
XY
D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000235:genetics* / Q000378:metabolism; D008263:Macrophage Migration-Inhibitory Factors / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020798:Two-Hybrid System Techniques
D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D008263:Macrophage Migration-Inhibitory Factors; D010446:Peptide Fragments; D011994:Recombinant Proteins; C045084:invariant chain