Identification of the interactions between the truncated fragments of macrophage migration inhibitory factor and CD74 using a yeast two-hybrid system.
- Author:
Zhi-xi SHAN
1
;
Qiu-xiong LIN
;
Chun-yu DENG
;
Hong-hong TAN
;
Su-juan KUANG
;
Ding-zhang XIAO
;
Jie-ning ZHU
;
Yong-heng FU
;
Xi-yong YU
Author Information
- Publication Type:Journal Article
- MeSH: Antigens, Differentiation, B-Lymphocyte; genetics; metabolism; Cloning, Molecular; Escherichia coli; genetics; metabolism; Extracellular Matrix; metabolism; Histocompatibility Antigens Class II; genetics; metabolism; Macrophage Migration-Inhibitory Factors; genetics; metabolism; Peptide Fragments; genetics; Protein Interaction Domains and Motifs; genetics; Recombinant Proteins; biosynthesis; genetics; Two-Hybrid System Techniques
- From: Journal of Southern Medical University 2009;29(12):2383-2390
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSBy 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.
RESULTSThe 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.
CONCLUSIONMIF interacts with the intact extracellular segment of CD74 (CD74(73-232)) independent of the functional domain of MIF(50-65).
