ConnectivetissuegrowthfactorbindingpeptidespecificExpressionandPurification

1、 connective tissue growth factor binding peptide-specific expression and purification 【abstract】 objective: using genetic engineering techniques to obtain and connective tissue growth factor (ctgf)-specific binding of small peptide. methods: the design and synthesis with an enterokinase cleavage sit

2、e of the gene fragment into pet 32 (a) plasmid vector downstream of thioredoxin gene, the recombinant plasmid into e.coli bl21 expression in bacteria, using a final concentration of 1mmol * l-1 of the iptg-induced expression of fusion protein; using thermal denaturation and ammonium sulfate fraction

3、ation of the initial fusion protein purified by affinity chromatography and then further purified by enterokinase cut, the final layer of gel filtration analysis of the purpose of isolated small peptides; by rp-hplc purity of the obtained identification of small peptides. results: the results of the

4、 recombinant plasmid was sequenced in line with expectations; induced expression later sds page identified, was found in the molecular weight of 20000 department has thick bands of expression appears in line with expectations; fusion protein thermal denaturation and a preliminary purification of amm

5、onium sulfate fractionation, were respectively 78.6% and 52.3%; purified with affinity chromatography the yield of 50.5%; by enterokinase after cutting were isolated by gel filtration chromatography the purpose of small peptides, with the anti - gc identification of its purity was above 95%; the fin

6、al purpose of 3.4mg per liter fermentation broth to obtain peptides. conclusion: the successful preparation of ctgf-specific binding peptides, in order to further study of the biological activity of small peptides basis. keywords: connective tissue growth factor-specific peptide separation and purif

7、ication connective tissue growth factor (connective tissue growth factor, ctgf) is a bradham et al 1 in 1991 for the first time in human umbilical vein endothelial cells, cytokines found in the medium, from 349 amino acids composition; mainly by the skin into the fiber cells 2, glomerular mesangial

8、cells 3, hepatic stellate cells 4, lung fibroblasts 5, vascular smooth muscle cells 6 such as the synthesis and secretion. ctgf can promote cell proliferation, synthesis of collagen-mediated cell adhesion 7 8, induce apoptosis 9 11, promote blood vessel formation 12, stimulate fibroblast growth, and

9、 involved in regulating cell differentiation, embryo development, as well as wound healing, in a variety of organs and tissue fibrosis, may play an important role in the development process. in this study, we used phage display technology previously found that ctgf-specific binding peptides, based o

10、n 13, the use of genetic engineering technology to prepare the small peptide, in order to further study its biological activity, developed into small-molecule inhibitor of ctgf basis. 1 materials and methods 1.1 materials bl21 (de3) hsds gal (cits857 indi sam7 nin5 lacuv5 t7i), jm109, the laboratory

11、-preserved; pet 32 (a) plasmid, enterokinase kit, novagen inc.; his selecttm, sigma company; sephadex g25, phamarcia company; restriction endonuclease bamh , xho , ecor , t4 dna ligase, promega corporation; gel extraction kit, the shanghai public health and bio-engineering technology co., ltd.; bio-

12、rad, beijing, 61 instrument factory; level shaker, taicang city, bright experimental analysis instrument factory. 1.2 methods 1.2.1 synthesis of the target gene according to our previously from a random 12-peptide library was screened phage clones obtained dna sequencing, synthesis of a pair of ente

13、rokinase sites, encoding gene (810a) for each pair of primers pa1 and pa2, with ddh2o dubbed 50pmol * l-1 primer solution. pcr reaction is as follows: dntp (10mmol * l-1) 0.5l, 10 buffer 2.5l, pa1 and pa2 of 1l, taq enzyme (10u * l-1) 0.2l, final ddh2o padded to 25l; pcr reaction conditions : 94 pre

14、degeneration 5min; 94 denaturation 30s, 60 annealing 30s, 72 extension 30s, 30 cycles; 72 extension of 7min, 4 insulation. pcr products were 20 g * l-1 agarose gel electrophoresis, after electrophoresis, eb stained 5min, uv lamp to observe and the purpose of cut bands, purified by gel extraction kit

15、 fragment. 1.2.2 construction of expression vector -containing pet 32 (a) plasmid of e. coli was inoculated with ampicillin (final concentration of 100g * ml-1) of the mh amp plates, 37 overnight train. single colonies were picked, inoculated with the same quality at a final concentration of ampicil

16、lin in lb medium, 37 zhenyao to a600 nm of 0.6, extract pet 32 (a) plasmid dna, 12g * l-1 agarose gel electrophoresis results were observed. with restriction endonuclease bamh and xho , 37 digested pet 32 (a) plasmid dna and the pcr product of 2h, 65 20min inactivated. 12 g * l-1 agarose gel electro

17、phoresis, after electrophoresis, eb staining, uv lamp to observe and cut out the purpose of bands, gel extraction kit for gel extraction. in the ep tube followed by adding recovery of plasmid dna10l, gene fragments 1l, 10 connection buffer 1l, t4dna ligase 1l, 4 overnight connection. connect the pro

18、duct into jm109 competent cells, incubate on ice 30min, 42 heat shock 90s, to join a preheated lb medium 1ml, 37 water bath, 30min, centrifuged to precipitate coating on containing ampicillin (final concentration of 100g * ml-1 ) mh amp plates, 37 overnight train. randomly selected a single colony w

19、as inoculated with ampicillin (final concentration of 100 g * ml-1) in lb liquid culture medium, 37 shaking 6h, extracted plasmids were digested with restriction endonuclease bamh , xho and ecor digestion , 12g * l-1 agarose gel electrophoresis results were observed. plasmid-positive health workers

20、sent to shanghai biological engineering technology co., ltd. sequencing, sequencing primers for the t7 terminator primers. 1.2.3 expression of fusion protein obtained recombinant plasmid dna 5l transformed bl21 (de3) competent cells, 37 incubation overnight, pick a single colony after amplification

21、of 1% of the inoculum adapter 250ml medium, 37 zhenyao to a600 nm of 0.6 0.8, add a final concentration of 1mmol * l-1 of the iptg, 37 induced by 8h. collected by centrifugation pellet was added to 12ml in the biomass containing 50mmol * l-1tris hcl, 1mmol * l-1 edta, 1mmol * l-1 pmsf, 320l 10g * ml

22、-1 lysozyme mixture, uniform stirring 30min, 15000r * min-14 centrifugal 15min, take the supernatant, placed in -20 to save. 1.2.4 isolation and purification of small peptide 1.2.4.1 thermal denaturation get broken supernatant, placed in 65 water heating, denatured hybrid protein, heat denaturation

23、of the time were 15,30,60,120 min, 12000r * min-1 centrifuge 5min later, taking on the clear liquid with sds page analysis, and calculate the yield of fusion protein. 1.2.4.2 ammonium sulfate fractionation of the fusion protein after heat denaturation at 25 for ammonium sulfate fractionation. select

24、 the saturation ammonium sulfate solution of 20%, 30%, 40%, 50%, 60% of the experiment; with 50mmol * l-1 tris hcl (ph8.5) dissolved after the detection of coomassie brilliant blue protein determination kit its protein content, sds page analysis used to calculate the yield of fusion protein. 1.2.4.3

25、 affinity chromatography to his selecttm affinity and gel loaded column with guanidine hydrochloride, edta, nickel sulfate affinity according to operating instructions and plastic recycling; two-fold volume of pbs with the balance of the sample by adding columns, flow rate control in 15ml * h-1 or s

26、o, with five times the volume of pbs washing, flow control in 100ml * h-1 or so. then with five times the volume, respectively 20,100,200 mmol * l-1 imidazole elution flow rate was controlled at about 60ml * h-1, collect elution peak collected in each tube 3ml. merger-eluting peak, with 0.01mol * l-

27、1 ph7.4 in pbs dialysis overnight, peg 20000 concentrated to about 2ml. 1.2.4.4 enterokinase enterokinase cutting a unit is defined as: at 20 , it will be 50g fusion protein completely cut the amount of enzyme required. press kit specification asked to join the right amount of protein, reaction buff

28、er and enzyme, sds page analysis of different time enterokinase cutting efficiency of the fusion protein. 1.2.4.5 gel filtration chromatography sephedex g25 gel chromatography of small peptides, elution of 0.1mol * l-1ph 7.4 in pbs, flow rate of 15ml * h-1, step by step collection, each tube to coll

29、ect 3ml , merge the last eluting peak, freeze-drying. 1.2.4.6 rp hplc identification of pure freeze-dried powder dissolved in distilled water with the right amount, 0.22m membrane filtered, using c18octadecyl rp column (bio rad) for analysis to contain 0.05% trifluoroacetic acid in acetonitrile wate

30、r mobile phase, acetonitrile concentration from 5% to 95% gradient elution, detection wavelength 225nm. 2 results 2.1 construction and identification of expression vector with the specificity of enterokinase site synthesized by pcr using primers 810a of the dna sequence encoding, pcr products recove

31、red by bamh xho restriction enzyme digestion, the product with the same enzyme digested pet 32 (a) the corresponding fragment connected with the t4 enzyme-linked transformed into e.coli jm109 after extracting plasmid dna, respectively bamh , xho , ecor digestion of single, 1.2% agarose gel electroph

32、oresis, bamh and xho recombinant plasmid can be cut, but no digestion of ecor (figure 1), suggesting that exogenous fragment inserted bamh and xho between sites; sequencing results in line with the expected sequence (sequencing the report omitted). 2.2 the expression of fusion protein recombinant pl

33、asmid was transformed into bl21 (de3) competent cells, pick a single colony amplified by 1mmol * l-1 induced by iptg 37 for 8h, with 12% sds page observe the protein expression. the results showed that about 20000 molecular weight protein expression appeared dense band consistent with the expected m

34、olecular weight (figure 2). 1.marker; 2. induction 8h when the total protein expression; 3. when the total protein expression induced 0h reposted elsewhere in the paper for free download center 2.3 purification of specific binding peptides bacterial supernatant was broken by the 65 heat denaturation

35、, with time, hybrid protein gradually reduced, while the fusion protein is almost unchanged. after 30min heating, hybrid protein is no longer changed significantly. with 30% 50% of ammonium sulfate for salting and found that 40% saturation when the integration of protein, most of precipitation, whil

36、e the other impurities within the concentration range of protein deposition in this small, determined by the coomassie brilliant blue protein content and gel electrophoresis analysis, thermal denaturation and salting-out rate of the fusion protein were 78.6% and 52.3% (chart omitted). his selecttm w

37、ith the right fusion protein affinity chromatography to purify the crude extracts showed that, with 200mmol * l-1 imidazole fusion protein can be eluted from the column by sds page detected protein purity of 86%, integration protein yield of 50.5%. with enterokinase to the purified fusion protein to

38、 be cut, with time, the fusion protein was gradually cut to 16h fusion protein is completely cut (figure 3). digestion products were purified using sephadex g25, collecting the last peak, the appropriate concentration for later rp hplc purity test, resulting in chromatograms show a single peak (figu

39、re 4), the sample purity of 95%. 3 discussion studies have shown that a variety of signaling molecules such as tgf , ang , vegf, et1, bmp, igf, etc., through smad, pkc, ros, jak, erk and other signaling pathways induced by the majority of glial elements, synthesis, inhibiting its degradation, and ca

40、n regulate the expression of integrin matrix receptors in tissues and organs resulting in fibrosis 14 20. in all of these signaling pathways activated pathway, ctgf is a key link in the occurrence and development of fibrosis play a key role in 21. therefore, ctgf is a more specific target site, scre

41、ening and preparation combined with ctgf-specific small peptide of great significance. the research and design a pair of encoding ctgf-specific primers binding peptides, while the upstream gene of the role of the introduction of enterokinase site (ddddk), was synthesized by pcr with enterokinase gen

42、e locus, and inserted through the bamh xho fusion protein (thioredoxin) downstream, so that when the purpose of protein expression, the use enterokinase cut, it will form a complete and mature small peptide. enterokinase role in the optimum ph value of 7.4, this experiment found that in the ph value

43、 of 7.4 under the conditions of digestion, the fusion protein will be a large number of precipitation, resulting in reduced efficiency of digestion. therefore, as an experiment, select the ph 8.0 digestion conditions, and adding a certain glycerol, groping at this ph value of enzyme cutting efficien

44、cy, and to determine the amount of enzyme under a certain time required for digestion. the results showed that the amount of enzyme in the standard, and as the extension of time, the fusion protein was gradually cut to 16h fusion protein was completely cut. to pet 32 (a) vector to express fusion pro

45、tein with a very good heat resistance at 90 above the water can be stabilized jian-cun 10min, this way, the fusion protein can be purified to remove bacterial broken solution, 60% 70 % of the hybrid protein, while denaturation of the bacterial protease broken solution to prevent protease fusion prot

46、ein hydrolysis. in this study, 65 condition of a more moderate thermal denaturation experiments showed that achieve the desired effect of the removal of miscellaneous proteins. although affinity chromatography is the most effective separation of proteins with his tag label the way, but its non-speci

47、fic adsorption and easily contaminated and reduced service life has been a constraining factor to their widespread use. in this study, affinity chromatography using ammonium sulfate fractionation prior to the fusion protein extracted from a complex solution out of the purity of the fusion protein in

48、creased more than 60% re-use affinity chromatography purification, this hybrid protein, reduced the pro-and chromatography resin pollution, while reducing the affinity of the non-specific adsorption, increasing the efficiency of affinity chromatography purification. gel filtration chromatography is

49、the separation of large molecular weight difference between the peptides or proteins is one effective method, the purpose of this study is relatively small peptide molecular weight of about 1400, so choose sephadex g25, its separation range of 1 000 to 5 000, the experimental results show that using

50、 this method can achieve the desired separation effect, purified by reverse-phase chromatographic analysis of the purpose of small peptide, its purity reached above 95%. thermal denaturation ammonium sulfate fractionation by affinity chromatography, gel filtration chromatography enterokinase cutting

51、 steps such as freeze-dried, after calculating the final availability of 3.4 mg per liter fermentation broth of the purpose of peptides. the success of the small peptide preparation for further research and ctgf binding activity, affinity constant and whether it can inhibit the biological activity o

52、f ctgf basis. 【references】 1 bradham dm, igarashi a, potter rl, et al. connective tissue growth factor: a cysteine rich mitogen secreted by human vascular endothelial cells is related to the src induced immediate early gene product cef 10 j. j cell biol, 1991, 114 (6): 1285 1294. 2 takehera k. growt

53、h regulation of skin fibroblasts j. j dermatol sci, 2000, 24: 70 77. 3 wahab na, yevdokimova n, weston bs, et al. role of connective tissue growth factor in the pathogenesis of diabetic nephropathy j. biochem j, 2001, 359: 77 87. 4 hayashi n, kakimuma t, soma y, et al. connective tissue growth facto

54、r is directly related to liver fibrosis j. hepatogastroenterology, 2002, 49 (43): 133 135. 5 kucich u, rosenbloom jc, herrick dj, et al. signaling events required for transforming growth factor beta stimulation of connective tissue growth factor expression by cultured human lung fibroblasts j. arch

55、biochem biophys, 2001, 395 (1) : 103 112. 6 fan wh, karnovsky m j. increased mmp 2 expression in connective tissue growth factor over expression vascular smooth muscle cells j. j biol chem, 2002, 277 (12): 9800 9805. 7 jedsadayanmata a, chen cc, kireeva ml, et al. activation dependent adhesion of hu

56、man platelets to cyr61 and fisp12/mouse connective tissue growth factor is mediated through integrin alpha (iib) beta (3) j. j biol chem, 1999, 274: 24321 24327. 8 chen cc, chen n, lau l f. the angiogenic factors cyr61 and connective tissue growth factor induce adhesive signaling in primary human skin fibroblasts j. j biol chem, 2001, 276: 10443 10452. 9 hishikawa k, oemar bs, nakaki t. static pressure regulates connective tissue growth factor expression in human mesangial cells j. j biol chem, 2001, 276: 16797 16803. 10 hishikawa k, n