Screening For Proteins Interacting With MT1H In Human Lung Cancer Library Using Yeast Two Hybrid System

Metallothioneins (MT) are a group of low-molecular weight, cysteine-rich intracellular protein, which are encoded by a family of genes containing at least 10 functional isoforms (including MT1A, MT1B, MT1E, MT1Q MT1F, MT1X, MT1H). MT includes 61 to 62 amino acids, MT family can be divided into MT1, MT2, MT3 and MT4 four kinds of isomers. MTs regulate the binding of heavy metals (zinc, etc.)and protein,they also regulate gene expression,cell growth, free radical scavenging and detoxification.Formerly worke mainly concentrated on the MT1H between it and some heavy metals (for example zinc, cadmium, copper, silver and so on) metabolism aspect, but the related MT1H interaction protein knows really few, we has not seen the related reports. There are few reports on the expression of the different isoforms of MT which have been analyzed by specific gene probes. They reveal that certain isoforms are expressed in specific cell types.Recently, downregulation of MT gene expression in PTC(papillary thyroid carcinoma)has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors., In wetting quality mammary gland drive pipe cancer MTⅠANDⅡare upregulation; At the digestive tract cancer, the testicle gonad tumor, the salivary gland tumor, the ovarian cancer, the lung cancer, the melanin lump, in the colon cancer organization observes the MT expression, the above has not carried on the division to MT in the oncology research to MT each hypotype, possible MT each hypotype to play the different role in the different tumor. A number of studies have shown an increased expression of MT in various human tumors of the breast, colon, kidney, liver, lung, nasopharynx, ovary, prostate, salivary gland, testes and urinary bladder. The above has not carried on the division to MT in the oncology research to MT each hypotype, each hypotype of MT possibly plays the different role in the different tumor.some reports reveal MTs production is related with the carcinogen stimulation and the cancer gene activation concerns,in the carcinogenic process.such as chemistry carcinogen ethyl armor nitrourea may activate in vitro raise S49 the cell and lead to MT incrensing. Then, which type of proteins has interaction with MT1H in the tumor,particularly in the lung cancer? We use the yeast double hybrid technology to screen initially related protein with the MT1H for further studying its function to provide the basis.Materials and Methods1,Library, strain, material particle and reagentMATCHMAKER GAI4 yeast two hybrid system3, Clontech:BD clone material particle pGBKT7, AD the clone material particle pGADT7, BD material particle masculine gender compares pGBKT7-T, the BD material particle negative compares pGBKT7-Lam. Yeast strain AH 109, yeast culture medium YPDA, the synthesized nutrition gap culture medium (synthetic dropout minimal base, SD) SD/-Trp, SD/-Leu, SD/-Trp/-Leu, SD/-Trp/-Leu/-His, SD/-Trp/-Leu/-His/-Ade, the X-a-glycosidase (X-a-gal) buys from Clontech Corporation. The lung cancer cDNA library buys from Clontech Corporation. Backwoods coli TopolOC is our working office preservation. Needs the restrictive interior contact enzyme, LA the TaqDNA polymerase, T4 the DNA ligase buys from TaKaRa Corporation. The acid pickling beaded glass buys from Sigma Corporation.2,Methods(1) Bait BD material particle's(pGBKT7-MT1H) construction and identification Obtain the MT1H gene of the human.use the double enzyme cuts the position after PCR the insert NdeI/BamHI.that clones to pGBKT7, transformation backwoods coli Topo10, Kanamycin select Positive clone, the double enzyme cuts extract the particle that identifies the insertion piece size, the insertion piece size is BD particle pGBKT7-MT1H, compare the result with the MT1H of GeneBank sequence about homology.(2) yeast double hybrid screening①Apply the lithium acetate method to make yeast two-hybrid system bait vector pGBKT7-MT1H reorganization and lung cDNA library plasmid DNA co-transformed into yeast strain AH 109, coated in SD/-Leu/-Trp/-His/-Ade/Xa-gal plates,30℃cultured colony grew, the blue screen positive clones. All single colony picked positive clones in the SD /-Leu /-Trp /-His /-Ade /Xa-gal plates, passaged three times repeatedly, Use Xa-gal each time filter to the exclude false positive.②Extracte E.coli plasmid DNA with pACT 5',3'AD primers PCR, products are HaeⅢdigestion, agarose gel electrophoresis, remove the duplicate clones. Plasmid DNA sequencing of the positive identification. The GeneBank bioinformatics analysis to determine gene.③Back cross validation:the positive clones are inoculated in SD/-Leu/-Trp/-His /-Ade liquid culture medium, the glass bead extraction of yeast. Positive clone plasmid was transformed into E. coli TopolOC, kanamycin select clones. plasmid DNA and the recombinant plasmid pBGKT7-MT1H yeast strains co-transformed into AH109. Inoculated the products on the SD/-Leu/-Trp/-His/-Ade/Xa-gal.If not growth or the growth of white colonies were ruled out as a false positive.Results1,the bait plasmid pGBKT7-MT1H Construction and identificationpGBKT7-MT1H by EcoRI/BamHI double-digested, resulting in about the size of DNA fragments of 242bp, and the theory of the same size was amplified. Sequencing results showed that the integration of the region and have the correct reading frame in the right direction. Confirmed by DNA sequencing,the result is no mutation.2,The bait plasmid pGBKT7-MT1H toxicity testing and self-activating effectThe diameter of the same AH 109 (pGBKT7-MT1H) and AH 109 (pGBKT7) were in 3ml of YPDA liquid medium overnight incubation,600nm absorbance of 1.20 and 1.22 respectively, showed that the constructed yeast bait plasmid hasn't toxicity and don't affect yeast normal growth. AH109 (pGBKT7-MT1H) in SD/-Trp/X-α-gal white colonies grown in SD/-His/-Trp/X-α-gal showed no growth, excluding the pGBKT7-MT1H ability to activate the reporter gene itself.3,Gene sequence and analysis(1) the classification of positive clonesIn SD/-Leu/-Trp/-His/-Ade/X-agal plate blue screen positive clones. False positive will be excluded after the plasmids were digested with HaeⅢendonuclease to 10 g/L agarose gel electrophoresis, can see the different sizes of cDNA fragments is divided into 11 categories according to fragment size.(2) sequencing resultsAccording to the fragment size of the classification results, we have selected eight kinds of proteins.ConclusionWe select eight kinds of proteins that interact with MT1H.