TF, 679 80 kd TFN TFC II TF PTH TFN TFN PCR PCR PTH TF pet22-b ppic9 BL21(DE3) GS115 SDS-PAGE N TFN N (PTH-TFN) GS115 SDS-PAGE ppic9-pth-tfn/gs115 40 mg/l 5 100 mg/l PTH-TFN TFN 90% Western Blotting PTH-TFN PTH PTH-TFN TFN PTH TFN Caco-2 - -
PTH-TFN TFN PTH - -
Expression,Purification and Bioactive Characterization of Transferrin and Its Fusion Protein Abstract The human transferrin TF is a metal-bingding glycoprotein of 679 amino acids and a molecular weight of 80 kd.the protein is arranged in two lobes(tfn,tfc) which are similar in character and both binds Fe.The fundamental role of transferrin is to control the levels of free iron in body fluid by binding and transporting Fe 3+ ions,which will help to provide the availability of iron and prevent the deposition of iron in the blood. The transferrin receptor is II type diglycoprotein which mainly appeared to be malignant cell,blood-brain barrier,overproliferation cell and small intestine. Many studies affirmed that insulin-transferrin conjugate can be taken in orally and reduced blood sugar.so we constructure Humane parathyroid hormone PTH -TFN to explore the possibility of its transportion across cell membrance while TFN as carrier Humane parathyroid hormone and sorts of human tansferrins gene were respective amplified by using PCR technique from conserved plasmid and composed combination gene by using overlap PCR technique.these genes were subcloned into eukaryotic expression vector-pet22-b and prokaryotic expression vector-ppic9 and then transformed into correspond strains E.coli BL21(DE3) and Pichia pastoris GS115. Transformants were screened by induction in shake flask.sds-page of the supernatant of - -
ppic9-pth-tfn/gs115 and ppic9-tfn/gs115, which result in highly expressing strains, showed that only TFN and PTH-TFN can expressed in pichia pastoris GS115.In shake flask the expression level of ppic9-pth-tfn/gs115 is about 40 mg/l, while in a 5 L fermentor the expression level reaches 100 mg/l. After partial purification, the purity of TFN and PTH-TFN exceeds 90%. Western blotting showed PTH-TFN could react with anti-human PTH monoclonal antiboby, Fe saturation study affirmed that TFN still can binded Fe.These comfirmed TFN and PTH-TFN still conserved their origial construction. Caco-2 cell origins from human colon adenocarcinoma,which monolayer cell often are applied as a model of administered drug in vivo. In this study it illustrated that PTH-TFN can be transcellar delivered, which reveal, as a transcellar carrier, TFN can help PTH absorbed into body across intestine Key words: Humane parathyroid hormone,transferrin, pichia pastoris gene expression - -
[1] [2] [3.4] [5] [6] [7.8] [9.10] P 97 [11] 670 700 80 kd, ph 5.6-5.8, ph 8.7 2.5 mg/ml 35 umol/l 0.2-0.3, 30% [12] [13] 1987 Anderson, [14] [15] X-, TFN TFC - -
, Fe 3+ Fe 3+ TFN TFC Fe 3+ TFN ph 5.5 Fe 3+ TFC ph 5.0 Fe 3+ Tyr 517 426 His 585 Asp 392 1 HCO 3 Asp 63 Tyr 188 Try 95 His 249 Fe 3+ Fe 3+, Fe 3+ Fe 3+ Fe 3+, ph, ATP H + ph 5.0 6.0 Fe 3+ Fe 3+ Fe 3+ [16] Fe 3+ 465nm [17] 38 Cys 19 8 TFN 11 TFC [18] TFC 431 611 Asn N [19] 3 Q21-25 [20] 42% TFN TFC 40% [21.22.23] Fe 3+,, - -
,,,, [24] [25.26] Transferrin Receptor TFR [25.27.28] TFR II 5.2 170 000-200 000 D 90 kd [29.30] 760 N 1-67 68-88 89-760 Tyr Thr(104) Cys(89 98) N 122-188 384-606 189-383 607-760 - [31] TFC [32] - -
TFC C1 TFN N1 [31] TFN TFR ph TF TFR TF Fe 2 -TF TF Fe 3+ TFR Fe 3+ - - [33] 6 [34] - - - ATP ph 5-6 [35] DMT1 [36.37] - ph [38] - -
1/3, [39],, [40], [41.42.43] - BeWO IEC6, [44.45] [46] -, [47],, - [48] DNA DNA - -
DNA, [49], [50], EMT-6 [51] K562 HL60 [52.53.54.55] KB-8-5 KB C1 KB-V1 L929 RT-4 [56.57.58.59.60] [61] KB-8-5 KB-C1 KB-V1, IC 50 [62] [63] DNA, HepG2 HL60 [64.65] DNA - -
,, DEAE- DNA DNA DNA DNA -Gal GFP (HSV-TK) K562 M7609 TMK-1 [66], myb HL60,,,, [67] CRM107 CRM107 60 [68] - -
Ali HIV-1 HIV-1 [69] PTH TF, TF PTH-TF TFN Caco-2 [70] PTH Caco-2 - -
2.1 2.1.1 GS115 DH5α BL21 (DE3) 2.1.2 ppic9 pet22-b 2.1.3 ATCC PTH 2.1.4 Caco-2 2.2 2.2.1 PTH HRP 2.2.2 - -
NotI XhoI BamHI BglII SacI PshBI pyrobest DNA rtaq DNA T 4 DNA 2.2.3 DNA Q.BIOgene Promega 2.2.4 Marker DL2000 DL15000 Marker 14.4 20.1 30.0 43.0 66.0 97.0 kd Pharmacia 2.2.5 Oxiod Oxiod YNB Difco IPTG Inaic Sigma 2.3 2.3.1 5PET: 5'-ATGCATTAATGGTCCCTGATAAAACTGT GA-3' PshBI 3PET: 5'-CGGATCCTAAGGTCTACGGAAAGTGCA GG-3' BamHI PET5: 5'-ATGCATTAATGGATGAATGCAAGCCT-3 PshBI - -
XhoI Pt3:5'-AAAGCTAAATCCCAGGGTGGCGGTGGCTCT GTCCCTGAT AAAA CTGTGA-3' Pt4:5'-AGAGCCACCGCCACCCTGGGATTTAGCTTTGGTC AGA-3' Ptc1: 5'-ATGCATTAATGTCAGTATCAGAGATTCA GTTA-3' PshBI Pt c3: 5'-AAAGCTAAATCCCAGGGTGGCGGTGGCTCTCC AACA GATGAATGCAAGCCT-3' 2.3.2 5Y: 5'-CCGCTCGAGAAAAGATCAGTATCAGAGATTCAGTTA ATG-3' XhoI 3Y: 5'-ATAAGAATGCGGCCGCTTAAGGTCTACGGAAAG TGCA GG-3' NotI 5YTF: 5'-CCGCTCGAGGTCCCTGATAAAACTGTGA-3' 3YTFn: 5'-ATAAGAATGCGGCCGCTTATTCGATCTGT TGGGGCTTCTGG-3' NotI YTFc5: 5'-CCGCTCGAGCCAACAGATGAATGCAAGC CT-3' XhoI XhoI 5YTFc: 5'-CCGCTCGAGTCAGTATCAGAGATTCAGTT-3' 2.4 BIOSTAT B 5 B.Braun Biotech TZ-2EH DYIII 2C - -
DYCZ-30 UNIC-UV2100 DNA thermal cycler T personal(biometra ) TGL-16C AKTA primer Pharmacia Millipore Millipore SHZMADZA DYY-III2 Chemcadet ph SAKURA SP Sepharose Fast Flow XK16 20 ( ) Phenyl Sepharose 6 Fast.Flow ( ) XK16 16 100 mm Pharmacia 2.5 LB 5 g/l 10 g/l NaCl 10 g/l ph7.0 LB 5 g/l 10 g/l NaCl 10 g/l ph7.0 10 g/l BMGY 1% 2% 1.34% YNB 4 10-5 % 1% 100 mmol/l ph6.0 BMMY 1% 2% 1.34% YNB 4 10-5 % 0.5% 100 mmol/l ph6.0 - -
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