Cao MengChang WeiweiXu YangFang LinjingLiu YuanGu Ning

（1School of Biological Science and Medical Engineering，Southeast University，Nanjing 210009）

（2Chien-Shiung Wu College，Southeast University，Nanjing 210096）

Construction and expression of GFP conjugated M IM-I-BAR

Cao Meng1Chang Weiwei1Xu Yang2Fang Linjing2Liu Yuan2Gu Ning1

（1School of Biological Science and Medical Engineering，Southeast University，Nanjing 210009）

（2Chien-Shiung Wu College，Southeast University，Nanjing 210096）

To achieve a visible inverse Bin-amphiphysin-Rvs（I-BAR）domain recombinantofm issing inmetastasis（M IM）protein，the green fluorescent protein（GFP）encoding gene was cloned at the terminal of M IM-I-BAR as a probe．The DNA was successfully constructed on a 6xHis-tagged prokaryotic expression plasmid．The non-GFP labeled M IM-IBAR encoding plasmid was also constructed as a control．Being successfully transformed into BL21（DE3）cells，the GFP-conjugated M IM-I-BAR（M IM-I-BAR-GFP）exhibits strong visible fluorescence，and the expression product can be easily detected by visual inspection，a fluorescence microscope，Western blot or ultraviolet and visible spectrophotometer．Moreover，examination of expression efficiency under various culture conditions revealed that the M IM-I-BAR-GFP gene has a high protein yield at 10℃，but not at the culture temperature of 37℃．This property ismuch different from that of the non-fluorescent M IM-I-BAR gene．This optimal expression condition is also proved to be feasible for protein production in midi-scale．The fluorescent recombinant M IM-I-BAR-GFP protein can serve as a useful tool in scientific research，biomedical application and pharmaceutical development．

missing in metastasis；inverse Bin-amphiphysin-Rvs；green fluorescent protein；plasmid；expression；purification

Plasma membrane plays a protective and functional role and it is vital for living eukaryocytes．Various cellular activities require membrane deformation，including polarization，endocytosis，filopodia／lamellipodia fomation，mobility，etc．，many of which are driven by intracellular membrane binding．Among these proteins，the Bin-amphiphysin-Rvs（BAR）fam ily is an importantThese kinds of proteins have the N-terminal BAR domain，forming a rigid anti-parallel dimer，b_inding to and deform ing themembranes into curvatures［56］．The inverse BAR（I-BAR）proteins are a subfam ily of BAR，forming a convex face and favor membrane protrusions rather than invaginations［7］．In the mammalian I-BAR fam ily，the m issing in metastasis（M IM）gene has attracted particular concerns due to its low expression in a subset of relatively higher metastatic potent bladder cancer cells［8］．The study of M IM protein can_ bring better understanding to certain human di_seases［915］and promote therapeutic research progress［1617］．The I-BAR domain is critical for the M IM function．It consists of 250 amino acids and binds to PI（4，5）P2-enriched membranes，shapesmembrane deformations by approaching the interior of tubules，interacts w ith small GTPase Rac，and cross-links actin．Recombinant M IM-I-BAR has been proved to be useful in many

To the best of our know ledge，there still remains a lack of proper methods to accurately identify M IM-I-BAR from other substances．A commercial antibody that specifically binds to M IM-I-BAR is so far unavailable．Even w ith a specific antibody，it is tedious to track，monitor or quantify the protein from bio-samples for the relatively inconvenient immunological assays．Chem ical labeling of M IM-I-BAR is also challenging since it is technically difficult to achieve and considerably expensive for purification．Therefore，directly labeling the protein which can make the preparation and detection more convenient is an urgent requirement．Considering that the green fluorescent protein is a useful probe for protein modification，in this paper we construct such plasm ids that can produce the M IM-I-BAR-GFP protein in the prokaryotic expression system by carrying genes encoding both M IM-I-BAR and GFP in a continuous sequence．We also find that the M IM-I-BAR-GFP gene under high expression conditions is quite different from M IM-I-BAR w ithout GFP．Finally，under the optim ized system，we prepare a remarkable amount of proteins and immobilize them on beads for storage aswell as for further applications．

1 Experimental

1.1 Chem icals and antibodies

A ll chem icals unless otherw ise indicated were purchased from Sigma Aldrich．The Ni-NTA resin was from Genscript．T4 DNA Ligase and restrict enzymes werefrom NEB．BL21（DE3）Competent E．coli and DNA agarose were from Invitrogen．The DNA Gel Extraction Kit was from AXYGEN．The DH5αCompetent E．coli，SDS-PAGE Gel Preparation Kit and Plasm id M idi Preparation Kitwere from Beyotime．Coomassie Brilliant Blue R-250 was from KeyGEN．Tryptone and yeast extract were from Oxoid．Pfu DNA polymerase was from Thermo Scientific．The 6xHis monoclonal antibody（clone 4A12E4）was from Lifetechnologies．

1.2 Plasm ids

The plasm id M IM-I-BAR-GFP used as a gene template was prepared as described previously［22］．Plasm ids encoding His tagged M IM-I-BAR or M IM-I-BAR-GFP proteins were prepared by ligation of PCR-generated DNA fragments into vectors pET-14b（Novagen）．DNA cloning was performed in the DH5αcells．The primers used in PCR are listed in Tab．1．

Tab.1 Designed primers for PCR

1.3 Recom binant proteins

Briefly，BL21（DE3）competent E．coli cells were transformed by the plasm ids，and the transformed cells were selected based on their resistance to ampicillin．A single colony of the transformed cells was added to 3 m L of Lennox broth（LB，1%tryptone，0.5%yeast extract and 1%NaCl，pH 7.4）medium containing 50μg／m L ampicillin and incubated at 37℃for 16 h at a shaking speed of 200 revolutions perminute．The culturewas further inoculated in 100 m L of pre-warmed LB medium w ith 50μg／m L ampicillin and shaking w ith a speed of 200 revolutions perm inute for another 1 to 2 h at37℃．When the optical density at the wavelength of 600 nm reached 0.3 or 0.4，the culture was added to w ith 0.5 mmol／L of isopropyl-beta-D-thiogalactopyranoside（IPTG）．A fter an additional 3 h of incubation，the cells were harvested in phosphate buffered saline（PBS），sonicated and centrifuged．The supernatant was incubated w ith Ni-NTA resin for2 h at4℃followed by three doses of wash w ith Buffer A（Sinopharm）and stored in Buffer A containing 0.02%NaN3．If bead-free proteins were needed，His-tagged proteinswere eluted with 250 mmol／L im idazole in Buffer A，and dialyzed using a Centricon YM-10 centrifugal filter（M illipore）．

1.4 Immunoblotting

After incubation，cellswere harvested by centrifuging，re-suspended in PBS，then sonicated and kept on ice before gel electrophoresis．A portion（50μL）of each sample wasmixed with 10μL 6x protein loading buffer（Beyotime），and 10μL of each m ixture was fractionated by SDS／PAGE，transferred to a nitrocellulosemembrane and subjected to Western blotting w ith anti-6xHis followed by horseradishperoxidase-conjugated secondary antibody in 100 mmol／L TBST（Tris／HCl，pH 7.5，150 mmol／L NaCl，and 0.05%（volume fraction）Tween20）containing 5%（mass fraction）non-fatm ilk powder．The antibody-reactive substances on the membrane were detected by the hypersensitive ECL detection kit（KeyGEN）．

1.5 Analysis of photoabsorption

Cells in liquid culturemedium were collected by centrifuging at 5 000 g．After washing and being re-suspended in PBS，the cellswere treated by sonication for lysis and then were kept in light resistant container at 4℃．Photoabsorption of the cellular proteins were analyzed by spectrophotometric measurement at 300 to 700 nm using the UV-3600 spectrophotometer（Shimadzu）．

2 Results and Discussion

The plasm id encoding His-tagged M IM-I-BAR-GFP was constructed as well as the control non-GFP plasm id pHis-M IM-I-BAR．Although GFP can act as a probe at both ends of the target protein，here we used GFP as a C-terminal tag tominim ize the effects of M IM-I-BAR dimer on the GFP C-term inal catalytic fluorescent group．W ith the plasm id M IM-I-BAR in pEGFP plasm id as the reaction template，M IM-I-BAR-GFP and M IM-I-BAR genes containing restricted clone sites were prepared by PCR using designed primers．As shown in Fig．1（a），PCR products were subsequently purified by gel extraction，and then were double digested by restriction enzymes to obtain the insertgenes．The enzymeswere NdeI／XhoI for M IM-I-BAR-GFP and XhoI／BamHI for M IM-I-BAR．The pET-14b vector was cut and purified in the meantime．Ligation was performed using T4 DNA ligase．The molar ratio of the insert gene and the vector gene is3∶1．a(chǎn)nd the geneswere then transformed into DH5αcells forselection under ampicillin．As shown in Fig．1（b），positive clones were collected，prelim inarily checked by double digestion（see Fig．1（c）），and further confirmed by deoxyribonucleic acid sequencing．The samples from left lane to right lane represent the standard markers，M IM-I-BAR-GFP and M IM-IBAR，respectively．The molecular size of the DNA bands are confirmed by comparing them w ith the standard markers，about 1.6 kilo base pairs（kbp）for M IM-I-BAR-GFP and about0.8 kbp for M IM-I-BAR．

Fig.1 The DNA examination in themolecular cloning procedures．（a）PCR；（b）Clones；（c）Verification

The vector plasm id pET-14b contains 6xHis-tag at the N-term inal of the cloned gene，which can be easily expressed in the BL21（DE3）prokaryocyte system and be purified w ith the aid of Ni ion beads．A fter sequence validation and m idi-preparation，the two type of plasm ids were successfully transformed into BL21（DE3）E．coli cells，which survived the selection of ampicillin on tissue culture plates．Interestingly，after being stored at a low temperature，clone cells carrying pHis-M IM-I-BAR-GFP plasm id displayed significantbackground GFP-linked protein expression as shown in Fig．2（a）．When suspended in the culture medium，the cells w ith green fluorescence can be easily observed under a fluorescent m icroscope（see Fig．2（b））．It not only suggests that the protein is capable of being expressed and correctly folded to obtain intrinsic functions，but also implies that the M IM-I-BAR protein tagged w ith GFPmay not necessarily express under the same condition that is suitable for cell grow th，which is around 37℃．

Fig.2 The strong green fluorescence of the transformed cells．（a）Clones on the culture plate；（b）Cells under fluorescentmicroscope

In order to obtain a relatively high protein yield，different temperature points including 10，20 and 37℃were exam ined for protein expression when culturing the transformed E．coliwith IPTG．The cell sampleswere collected and subjected to Western blot analysis．Since the pET-14b vectormarks each encoded protein a His-tag，the expression products can be detected and quantified using an antibody against His-tag by immunoblotting assay such as Western blot．

The non-fluorescent protein expression under different temperatures was first tested．Western blot using anti-6xHis antibody demonstrated that when recombinant His-M IM-I-BAR is expressed in BL21（DE3）under different temperatures，expression efficiency can be varied．At 37℃，the expression level is the highest，the quantification of which is about 25 fold than those at 10℃．However，for GFP-labeled M IM-I-BAR proteins，the expression efficiency is quite different from non-GFP M IM-I-BAR towards temperature alternation．Western blot results in an opposite expression-efficiency trends compared to nonfluorescent M IM-I-BAR．At 10℃，cell lysate contains significant larger amounts of His-M IM-I-BAR-GFP protein that develops a strong and clear band．But protein quantity decreased when the cells are cultured at 20℃，and the expression is almost fully compromised at 37℃（see Fig．3）．Samples from the left lane to right lane represent the standard molecular weight marker，cells cultured at 10℃，cells cultured at20℃，and cells cultured at 37℃，respectively．The molecular weight of the protein bands kilo Daltons（kDa）are confirmed by comparing them w ith the standard marker（about 28 kDa for His-M IM-I-BAR and about54 kDa for His-M IM-I-BARGFP）．

Fig.3 Temperature analysis for protein production．（a）Protein w ithout GFP label；（b）Protein w ith GFP label

The results can also be directly visualized by the clear fluorescent from the E．coli itself，ow ing to the advantage of the GFP protein probe．A fter being precipitated by centrifuging，pellets of the 10℃cultured cells system show a much brighter green color than those cultured at higher temperatures．Cells develop fluorescence under ultraviolet light，and the fluorescent level decreases remarkably when the incubation temperature goes up．The different light absorbance of the fluorescent protein was implemented using a spectrophotometer．The light absorption of the cell lysates features single peak around 485 nm due to the conjugation to a GFP-tag（see Fig．4），theintensity of which can be regarded as a measurement of the quantity of the protein．In accordance w ith the Western blot result，M IM-I-BAR-GFP recombinant protein produced at a low temperature gains higher light absorption．Considering that the absorbance is basically proportional to the concentration of the GFP-labeled protein，it confirms that10℃should be the optimal expression for M IM-I-BAR-GFP recombinant protein preparation．

Fig.4 The cellular GFP absorbance

After the bio-synthesis featurewas characterized，amidi-preparation was carried out under the optim ized condition to validate the productive consistency of the enlarged preparing system and the Ni-NTA binding ability of the His-tag product．A fter the cell culture volume was raised from 3 to 150 m L，the expression yield of the M IM-IBAR-GFP protein was still considerably high．These protein products are capable of being immobilized／collected using Ni-NTA beads（see Fig．5）．

Fig.5 M idi-preparation of the proteins．（a）M IM-I-BAR-GFP protein；（b）M IM-I-BAR protein

The beads binding proteins can be stored stably at4℃in light-proof containers for a certain time．Immobilizing the proteins also prevents them from degrading．Moreover，immobilized proteins can be directly used in pulldown experiments．If beads-free protein is needed，an elution using im idazole and a dialysis against proper buffer should be carried out，and an HPLC process is recommended for further purification．

3 Conclusion

Them issing in metastasis protein is a popular bio-macromolecule，particularly in the I-BAR domain，which is useful in scientific research and medical applications．We previously reported M IM-I-BAR recombinants construction usingHowever，GST is a relatively large protein tag that brings steric hindrance to a M IM-IBAR structure，and thus usually requires complicated cleavage procedure．His-tag is small and hasm inor influence on the conjugated protein［20］，which ismore suitable for M IM-I-BAR-GFP production．In this study，we amplified a M IM-I-BAR insert gene encoding a green fluorescent protein probe at the term inus．We found that although being cloned into the same vector pET-14b，genes w ith or w ithout a fluorescent probe encoding sequence displayed a direct opposite trend in expression efficiency w ith environmental temperature change．From 10 to 37℃，M IM-I-BAR protein yields increase as expected．Interestingly，the M IM-I-BAR-GFP protein expression is most efficient at 10℃，attenuated at 20℃and mostly comprom ised at 37℃，opposite to the temperature response of the non-GFP protein．Thismay be due to the toxic effects of GFP gene or its transcription products that potentially act intrinsically on the host BL21（DE3）cells at the higher temperature．Moreover，it is known that GFP fluorescence initiates better at a relatively low temperature［23］．Thismay be another reason for the different production efficiency between M IM-I-BAR constructs w ith or w ithout GFP．

W ith an optimal production temperature established，both proteins have been prepared inmidi-scale and roughly purified on Ni-NTA columns，which are used to store for future protein interaction experiments or for the sophisticated purification of beads-free proteins．The GFP probe is advantageous because it favors the M IM-I-BAR protein w ith a visible marker，so that fast and accurate analysis applications are supported，such as fluorescent m icroscopy，flow cytometry and m icroplate system．Therefore，these proteins are useful for studies involving M IM-I-BAR／membrane interaction，biomaterial modification，I-BAR dimerization and targeting peptide／compound development．

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綠色熒光蛋白探針標(biāo)記的M IM-I-BAR基因構(gòu)建和表達(dá)

曹 萌1常維維1許 陽2方琳靜2劉 袁2顧 寧1

（1東南大學(xué)生物科學(xué)與醫(yī)學(xué)工程學(xué)院，南京210009）
（2東南大學(xué)吳健雄學(xué)院，南京210096）

為制備可視化的轉(zhuǎn)移消失蛋白（M IM）的I-BAR結(jié)構(gòu)域重組體，克隆了順聯(lián)綠色熒光蛋白（GFP）探針編碼序列的M IM-I-BAR基因．在6xHis標(biāo)簽原核表達(dá)質(zhì)粒上成功構(gòu)建了DNA序列．同時(shí)，實(shí)現(xiàn)了未標(biāo)記熒光探針基因的M IM-I-BAR質(zhì)粒的構(gòu)建以作實(shí)驗(yàn)對照．成功轉(zhuǎn)染至BL21（DE3）大腸桿菌細(xì)胞后，GFP偶聯(lián)的M IM-I-BAR（M IM-I-BAR-GFP）蛋白表現(xiàn)出很強(qiáng)的可視熒光，該表達(dá)產(chǎn)物可方便的通過目測、熒光顯微鏡、免疫印跡和紫外可見分光光度計(jì)等多種手段進(jìn)行檢測．此外，在考察不同條件下的蛋白表達(dá)效率過程中發(fā)現(xiàn)，帶有GFP探針的M IM-I-BAR重組蛋白在溫度為10℃時(shí)產(chǎn)率最高，而并非37℃．這一特征與非熒光標(biāo)記的M IM-I-BAR明顯不同．研究證實(shí)該最佳表達(dá)溫度條件適用于重組蛋白產(chǎn)品中量制備．所開發(fā)的帶有熒光探針的M IM-I-BAR蛋白產(chǎn)品及其制備工藝在科學(xué)研究、生物醫(yī)學(xué)應(yīng)用以及藥物開發(fā)過程中均有較高的應(yīng)用價(jià)值．

轉(zhuǎn)移消失蛋白；inverse Bin-amphiphysin-Rvs；綠色熒光蛋白；基重組因；表達(dá)；純化

Q816

10．3969／j．issn．1003－7985．2015．03．009

2014-12-22．

Biographies：Cao Meng（1983—），male，doctor；Gu Ning（corresponding author），male，doctor，professor，guning＠seu．edu．cn．

s：The National Basic Research Program of China（973 Program）（No．2011CB933503），the National Natural Science Foundation of China for Key Project of International Cooperation（No．61420106012），China Postdoctoral Science Foundation（No．2013M541592）．

：Cao Meng，Chang Weiwei，Xu Yang，et al．Construction and expression of GFP conjugated M IM-I-BAR［J］．Journalof Southeast University（English Edition），2015，31（3）：353- 357．

10．3969／j．issn．1003－7985．2015．03．009