Mussel-inspired Binding Vascular Endothelial Growth Factor By Sortase-mediated Reactions To Modify Biomaterials

Many metallic materials are employed for medical devices.Surface modification of materials used for biological applications are usually required to provide suitable material surface environments for contact with cells and tissues.Although immobilization of growth factors is a method widely used for surface modification of medical devices,the modification methods are limited.Generally,silane coupling methods have been employed for the surface modification.However,recently a biomimetic approach inspired by mussel adhesive activity has been devised in addition to the conventional methods.Mussel secretes underwater adhesive protein,and 3,4-dihydroxyphenethylamine(DOPA)is known to be included in the active site.DOPA or more simplified one dopamine is employed for surface modification.Therefore,DOPA or the derivative dopamine has been used for modification of various materials including metals.Vascular endothelial growth factor(VEGF),one of the growth factor proteins,works as a key regulator for vasculogenesis and angiogenesis.We can expect that immobilization of VEGF on the medical materials promotes engraftment of the materials upon implantation.For this purpose,we tried to immobilize VEGF on medical materials made of cobalt-chromium(Co-Cr)alloy and titanium(Ti).Here,we attempted to incorporate DOPA into growth factor protein,vascular endothelial growth factor(VEGF),to add binding property onto metal surfaces.Since DOPA is a non-canonical amino acid,it is impossible to incorporate it into proteins by conventional protein engineering.Therefore,here protein ligation enzyme sortase A were employed as a bioorthogonal approach to modify the protein.The prepared VEGF had binding ability to metals and biological activity to moderately enhance the cell growth.For the immobilization of VEGF,we were inspired by the adhesive mechanism of mussels in which 3,4-dihydroxyphenethylamine(DOPA)plays a crucial role in adhesion.Thus,DOPA-containing adhesive peptides were intended to introduce into VEGF.1.Gene construction of p ET15b-VEGFTwo kinds of recombinant VEGF were prepared.The one is that containing his-tagged sequence at N-terminal recombinant VEGF(His6-VEGF),the other one is that carrying his-tagged sequence at N-terminal and sortase recognition site at C-terminal recombinant VEGF(His6-VEGF-Sor).The expression plasmid for His-tagged VEGF was construct as follows: DNA sequence encoding human VEGF 121 isoform(amino acid No.27 to 147)with his-tagged sequence at N-terminal was obtained by PCR using following primers;forward: 5'-GCCGCGCGGCAGCCATATGGCGCCGATGGCG-3';reverse: 5'-TATCATCGATAAGCTTGGGTTAACGACGCGGTTT-3';For his6-VEGF-Sor,DNA sequence encoding human VEGF 121 isoform(amino acid No.27 to 147)with sortase A recognition sequence(LPETGG)at carboxyl-terminal was obtained by PCR using a plasmid p FNCBD-VEGF as the template and following primer set;forward :5'-GCCGCGCGGCAGCCATATGGCGCCGATGGCG-3' and reverse 5'-AGCTTATCATCGATATGGGTTAACCACCAG-3'.The PCR amplified DNA was digested with Nde I and Hind III(NEB),and inserted into p ET15 b vector(NEB).After confirming DNA sequence,the expression vector was introduced into E.coli stain BL21(DE3)(Invitrogen).2.Recombinant expression of VEGFOne colony of BL21(DE3)E.coli include confirmed construct inoculated into TB Broth modified medium(Fluka,Sigma).The E.coli in log phase(OD600nm=0.6-0.8)was induced with 0.4 m M of IPTG and incubated at 37?,130 rpm,overnight.Expression was confirmed with SDS-PAGE was performed with 20% polyacrylamide gel(DRC).3.Recombinant VEGF purification and refoldingAfter induction by IPTG,the cell bodies were collected by centrifuging,8000 rpm,30mins,4?.Sonication,power output 3-4,duty cycle 30%-40%,5s,in sonication buffer(50m M Tris-HCl,1m M EDTA,100 m M Na Cl,2%Triton X-114),3times,then use contain 50 m M Tris-HCl,1m M EDTA,100 m M Na Cl wash 3 times.The cell bodies were resuspend in binding buffer(100m M Na H2PO4,6M Gua-HCl,5m M ?-ME,15 m M Tris-HCl,p H 8.0),and rotating for 30 mins,4?.After that,centrifuge,8000 rpm for 30 mins,take the supernatant then load into nickel affinity column(His Trap HP,GE.Healthcare).The recombinant protein was eluted by elution buffer(100m M Na H2PO4,6M Gua-HCl,5m M ?-ME,15 m M Tris-HCl,1M imidazole,p H 8.0).SDS-PAGE check the fractions,the collect the purified fractions,drop into the refolding buffer(500m M Tris-HCl,200 m M Na Cl,1m M EDTA 0.2m M GSSG,1m M GSH,1 M L-Arginine,p H8.0)to refold.Then use 3k Da membraneto concentrate protein.Finally,the recombinant protein was confirmed by 20% SDS-PAGE(CRP)and western blot.For western blot,the protein bands were transferred onto PVDF membrane(Amersham hybond-p GE.Healthcare).Then,the membrane was locked with 4% non-fat milk for 1 hour at room temperature.Then the membrane was incubated with rhuman anti-h VEGF antibodies solution(1/1000)at 4?,overnight.TBS-T was used to wash membrane,5min,3 times.Next step,using the goat anti mouse antibodies to incubated for 1 hour at 25?.After TBS-T washing 3 times,detected by ECL prime western blotting detection reagent(GE.Healthcare).Concentration of the purified protein was measured by ELISA for VEGF(R&D,quantikine kit).The purified protein in a freeze dryer system at-75? and stored at-80? for further use.4.Preparation of VEGF ligated with DOPA peptideThe synthetic scheme for VEGF ligated with DOPA-containing peptide XKXKX(GGGXKXKXGGGHHHHHH)peptide were synthesized by Bio-Materials Analysis Research Resources Center at the RIKEN Brain Science Institute.Sortase-mediated ligation of VEGF and DOPA-containing or control peptides were performed as 10 ?M VEGF,200 ?M XKXKX peptide,1 ?M Sa Srt A,1X Sortase Buffer,10 X Sortase buffer contain 500 m M HEPES(p H7.0),1.5M Na Cl,5.Characterization of Conjugated VEGFMass spectroscopy measurements were performed using an Applied Biosystems QSTAR Elite(Life Technologies,Carlsbad,CA,USA)for MALDI-TOF-MS,Circular dichroism(CD)measurements were performed using a spectrophotometer J-720(JASCO,Tokyo,Japan).Sample volumes of 250 ?L(100?g/ml)were measured using a 1 mm cuvette(scan type: continuous;scan speed: 100 nm/min;response time: 2.0 sec;bandwidth: 1.0 nm).Each sample was measured twice and the average spectra were obtained.Turbidity measurements were performed using 100 ?L of sample(1mg/ml)on a spectrophotometer V-550(JASCO,Tokyo,Japan)at a wavelength of 500 nm.6.Binding AssayThe binding affinities of the different VEGF derviatives towards titanium were studied by quartz crystal microbalance with dissipation monitoring(QCM-D).1?g/ml,5?g/ml,25?g/ml,and 50?g/ml VEGF-S,VEGF-Y,VEGF-D binding affinities were measured;10?g/ml VEGF-D in different p H(p H 4.5,p H 7.0,and p H 8.5)binding affinities were measured.7.Cell CultureHUAECs maintained in EGM2 medium at 37°C in 95% humidified air/5% CO2 were recovered by trypsinization and re-suspended in the medium for the following experiments.For the cell growth assay,HCAECs were recovered in a basal culture medium(EBM2,Lonza,USA)with 1% FBS but without angiogenic factors.The cell suspension was added to plates.The culture media was changed every 2 days,and after 5 days of culture,cell proliferation was assessed by a cell counting kit,WST8(Dojindo,Kumamoto,Japan).Conclusions: We introduced the sortase recognition site into the C-terminal of VEGF by the conventional recombinant DNA technique.At the same time,the adhesive DOPA containing peptide with oligoglycine attached in the N-terminal was synthesized by solid phase synthesis method.By the ligation with the sortase A,the adhesive peptide was ligated in the C-terminal of VEGF(VEGF-D).The binding affinity of VEGF-D onto the plates of Co-Cr alloy and Ti were measured by the quartz crystal microbalance(QCM-D)device.The results show VEGF-D has higher binding affinity than wild type.The bioactivity of VEGF-D was assayed with human umbilical vein endothelial cells(HUVECs).As a result,we confirmed that VEGF-D immobilized on the plates promoted cellular growth more than not immobilized one.