Protein Expression and Purification

Protein Expression and Purification 60 (2008) 221–224

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Protein Expression and Purification j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y p r e p

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli Yanling Hao 1, Xinyi Huang 1, Xiaohong Mei, Ruoyu Li, Zhengyuan Zhai, Sheng Yin, Ying Huang, Yunbo Luo * Col­lege of Food Sci­ence and Nutri­tional Engi­neer­ing, China Agri­cul­tural Uni­ver­sity, 17 Qing Hua East Road, Hai Dian Dis­trict, Bei­jing 100083, PR China

a r t i c l e

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Article history: Received 4 March 2008 Received in revised form 14 April 2008 Available online 26 April 2008 Key­words: Pec­tin methy­lest­er­ase inhib­i­tor Esch­e­richia coli Expres­sion Puri­fi­ca­tion Activ­ity anal­y­sis

a b s t r a c t A sig­nif­i­cant prob­lem in pro­duc­tion of fruit juices for human con­sump­tion is auto-clar­i­fi­ca­tion, where enzyme cat­a­lyzes pec­tin demeth­yl­a­tion result­ing in loss of the ‘‘nat­u­ral” cloudy appear­ance of juices. To over­come this prob­lem, a plant inhib­i­tor pro­tein which blocks the action of pec­tin methy­lest­er­ase has been used. In this paper, expres­sion of recombinant kiwi pec­tin methy­lest­er­ase inhib­i­tor (PMEI) was car­ried out in Esch­e­richia coli, and the tar­get pro­tein was expressed in the form of inclu­sion bodies. The expres­sion level reached 46% of total cell pro­tein. Then the fusion pro­tein was puri­fied by nickel ion metal affin­ity chro­ma­tog­ra­phy, and the purity was finally up to 98%. After refold­ing in GSH/GSSG redox sys­tem, recombinant PMEI not only could effi­ciently inhibit PMEs from eight dif­fer­ent plants, but could remain effec­tive inhib­i­tor activ­ity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential appli­ca­ tion in the pro­duc­tion of fruit juices prod­uct indus­try. © 2008 Published by Elsevier Inc.

One of the main prob­lems in the fruit prod­uct indus­try is the main­te­nance of the tur­bid­ity in fruit and veg­e­ta­ble juices dur­ing the pro­cess and stor­age. Pec­tin demeth­yl­a­tion by endog­e­nous pec­ tin methy­lest­er­ase (PME; EC3.1.1.11)2 is con­sid­ered as the main cause of cloud loss [1]. Now, in order to over­come cloud loss, the ther­mal treat­ment is widely used to inac­ti­vate PME in juice man­u­ fac­tur­ing. But the ther­mal pro­cess will severely dete­ri­o­rate nutri­ tional ingre­di­ents and sen­sory attri­butes of juices. In 1990, Bale­stri­eri et al. found a pec­tin methy­lest­er­ase inhib­i­tor (PMEI) in ripe kiwi fruit [2], which com­prises 152 amino acid res­i­ dues, account­ing for a molec­u­lar weight of 16.277 kDa [3]. Through form­ing 1:1 non-cova­lent com­plex to cover active site, the kiwi PMEI can com­pletely inhibit plant PMEs activ­ity [4]. Com­pared with ther­mal inac­ti­va­tion of PMEs, such milder treat­ment could remain bet­ter fla­vor qual­ity and higher vita­min con­tent [5]. Casst­ aldo et al. suc­cess­fully uti­lized the kiwi PMEI to pro­long orange juice shelf life to 9 months at 5 °C [6]. In addi­tion, affin­ity chro­ma­ tog­ra­phy on resin-bound PMEI can be used to con­cen­trate and detect resid­ual PME activ­ity in fruit and veg­e­ta­ble prod­ucts [7,8]. The dis­cov­ery of kiwi PMEI pro­vided a new way to solve fruit juice cloud loss. But, so far, the PMEI can only be obtained from kiwi fruit. Fur­ther­more, the extract­ing yield from kiwi fruit is

rather low. This greatly lim­ited the appli­ca­tion of PMEI in the juice indus­try. In order to con­ve­niently obtain PMEI, gene engi­neer­ing strain was taken into account. To date, E. coli has been widely used as the host strain to pro­duce het­er­ol­o­gous pro­teins due to its rapid growth rate, con­tin­u­ous fer­men­ta­tion capac­ity and rel­a­tively low cost [9]. Con­se­quently, in this work, we attempted to pro­duce recombinant PMEI on a large scale using E. coli expres­sion sys­tem. Mate­ri­als and meth­ods Mate­ri­als Esch­e­richia coli DH5a was obtained from Tian­Gen (Bei­jing, China). E. coli BL21 (DE3) and vec­tor pET-30a (+) that pro­duces a fusion pro­tein with His-Tag, was pur­chased from Nova­gen (Mad­ i­son, USA). Plas­mid pPIC9K/kWP­MEI con­tain­ing kiwi fruit PMEI gene was con­structed as described pre­vi­ously [10]. T4 DNA ligase and restric­tion endo­nu­cle­ases were pur­chased from Ta­Ka­Ra (Dalian, China). Cit­rus pec­tin (70% methy­leste­ri­fied), iso­pro­pylb-d-thio­ga­lac­to­py­ra­no­side (IPTG) and ruthe­nium red dye were obtained from Sigma (St. Louis, MO). Con­struc­tion of fusion expres­sion vec­tor

* Cor­re­spond­ing author. Fax: +86 10 62736479. E-mail address: [email protected] (Y. Luo). 1 These authors con­trib­uted equally. 2 Abbre­vi­a­tions used: PME, pec­tin methy­lest­er­ase; PMEI, pec­tin methy­lest­er­ase inhib­i­tor; E. coli, Esch­e­richia coli; IPTG, iso­pro­pyl-b-d-thio­ga­lac­to­py­ra­no­side; GSH, l-Glu­ta­thi­one (reduced); GSSG, l-Glu­ta­thi­one (oxi­dized); SDS–PAGE, sodium dode­ cyl sul­fate–poly­acryl­amide gel elec­tro­pho­re­sis; LB, Luria–Ber­tan­i. 1046-5928/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.pep.2008.04.004

The cDNA encod­ing kiwi fruit PMEI in the recombinant plas­mid pPIC9K/kWP­MEI was cloned into the fusion expres­sion vec­tor pET30a (+) as a Eco­RI–NotI frag­ment. The recombinant plas­mid, des­ig­ nated as pET30a-PMEI, was screened in DH5a. Then pET30a-PMEI was trans­formed into the E. coli strain BL21 (DE3) by heat shock.

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Expres­sion of the PMEI in E. coli

Results

A sin­gle-col­ony trans­form­ant was inoc­u­lated into LB medium con­tain­ing 50 lg/mL kana­my­cin and grown at 37 °C. The over­ night cul­ture was 50-fold diluted to inoc­u­late in 100 mL fresh LB medium. When the opti­cal den­sity (OD600) reached about 0.6, IPTG was then added to a final con­cen­tra­tion of 1 mM to induce expres­sion of fusion pro­tein for 4 h at 37 °C [11]. Cell pel­lets har­ vested were sus­pended in 1 mL PBS buffer (pH 7.4) and were sub­ jected to son­i­ca­tion in ice bath at 300 W with 10 s on/off cycle to pre­vent over­heat­ing, for a total time of 6 min. The total cel­lu­lar pro­teins were then par­ti­tioned into sol­u­ble and insol­u­ble frac­ tions by cen­tri­fu­ga­tion at 12,000g for 10 min at 4 °C. In order to deter­mine the sol­u­bil­ity of fusion pro­tein, the sam­ples were ana­ lyzed by SDS–PAGE.

Con­struc­tion of the fusion expres­sion vec­tor

Puri­fi­ca­tion and refold­ing of recombinant PMEI The inclu­sion bodies in insol­u­ble frac­tion were washed twice with buffer con­tain­ing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl and 0.5% (v/v) Tri­ton X-100. The washed inclu­sion bodies were fur­ ther dis­solved in bind­ing buffer con­tain­ing 20 mM sodium phos­ phate, 0.5 M NaCl, 20 mM imid­az­ole, 20 mM b-ME and 8.0 M urea (pH 7.4). After sol­u­bi­li­za­tion, the recombinant pro­tein was puri­ fied by affin­ity chro­ma­tog­ra­phy. Ni sepharose 6 Fast Flow media was packed and equil­i­brated with bind­ing buffer. The tar­get pro­ tein was eluted with a step­wise gra­di­ent of imid­az­ole from 20 to 200 mM. Puri­fied pro­teins were refolded by urea gra­di­ent dial­y­sis at 4 °C in PBS buffer con­tain­ing 1 mM EDTA, 1% gly­cine, 5% glyc­ erol, 0.9 mM GSH, 0.1 mM GSSG. Mean­while, urea was added into the PBS buffer every 12 h at a gra­di­ent con­cen­tra­tion (6, 4, 2, 1, 0.5 and 0 M).

The whole encod­ing region of kiwi fruit PMEI was cloned into the fusion expres­sion vec­tor pET-30a to gen­er­ate the recombinant plas­mid pET30a-PMEI. The deduced amino acid sequence of recombinant PMEI showed 98% homol­ogy with the native PMEI which was puri­fied from kiwi fruit. There were two dif­fer­ent amino acids (63 Thr ! Ala and 123 Asn ! Asp) between the native PMEI and recombinant PMEI. Expres­sion and puri­fi­ca­tion of PMEI The expres­sion of fusion pro­tein was induced with 1 mM IPTG at 37 °C for 4 h. After son­i­ca­tion and cen­tri­fu­ga­tion of bac­ te­ria cells, the sol­u­ble and insol­u­ble frac­tions were ana­lyzed by SDS–PAGE. The results showed that recombinant pro­tein was only found in the insol­u­ble frac­tion (Fig. 1), which indi­cated that the recombinant pro­teins were expressed in the form of inclu­ sion bodies. SDS–PAGE anal­y­sis revealed that the tar­get pro­tein accu­mu­lated up to 46% of the total cell pro­tein. After nickel ion metal affin­ity chro­ma­tog­ra­phy, the purity of the recombinant pro­teins reached 98% (Fig. 1). In order to pro­vide evi­dence for cor­rect refold­ing of the recom­bi­na­tion pro­tein, in vitro activ­ity of the PMEI were tested. In the exper­i­ment, puri­fied pro­teins refolded in PBS buffer con­tain­ing only 1 mM EDTA, 1% gly­cine and 5% glyc­erol showed no bio­log­i­cal activ­ity. How­ever, when 0.9 mM GSH and 0.1 mM GSSG were added into the refold­ing buffer, the recombinant PMEI could effi­ciently inhibit PMEs from eight dif­fer­ent plants. So, the pre­sen­ta­tion of the reduc­ing and oxi­diz­ing agents (GSH/GSSH) is essen­tial for PMEI inclu­sion body refold­ing.

Bio­log­i­cal activ­ity assay of recombinant PMEI The bio­log­i­cal activ­ity of recombinant PMEI was mea­sured in vitro accord­ing to its inhib­i­tory effect on PMEs from eight dif­fer­ent plants (tomato, orange, car­rot, apple, pine­ap­ple, haw­thorn, grape and kiwi). Plant fruit PME extrac­tion was prepared accord­ing to Giov­ane et al. [12]. Inhib­i­tory capac­ity of puri­fied recombinant PMEI to PMEs from dif­fer­ent plants was mea­sured using a gel dif­fu­ sion assay [13]. Gel con­tain­ing 0.1% pec­tin sub­strate was prepared at pH 7.0. Wells were punched in the gel. Excess amount of puri­fied recombinant PMEI was, respec­tively, mixed with a cer­tain amount of PME crude extrac­tions at room tem­per­a­ture for 15 min. Then, the mix­tures were placed into each well, and the whole well was sealed and incu­bated at 30 °C for 16 h. After incu­ba­tion, the gel was stained with 0.05% (w/v) ruthe­nium red dye for 45 min. Mean­ while, the same amount of the PMEs from eight fruits was respec­ tively assayed using the same method with­out any recombinant PMEI.

Inhib­i­tory effect of puri­fied recombinant PMEI on PME from dif­fer­ent plants The inhib­i­tory capac­ity of recombinant PMEI to PMEs was mea­ sured using a gel dif­fu­sion assay. The results showed that stained zones were around the wells con­tain­ing PMEs, indi­cat­ing that pec­tin was deme­thy­lat­ed by the PMEs from eight dif­fer­ent plants. When recombinant PMEI was incu­bated with PMEs, stained zones dis­ap­peared. The results indi­cated that recombinant PMEI could inhibit the activ­i­ties of PMEs from tomato, orange, car­rot, apple, pine­ap­ple, haw­thorn, grape and kiwi (Fig. 2).

Effects of pH and tem­per­a­ture on recombinant PMEI activ­ity Excess amount of tomato fruit PME crude extract was mixed with refolded recombinant PMEI and the mix­tures were pre-incu­ bated for 15 min (22.5 °C) at dif­fer­ent pH rang­ing from 3 to10. Mean­while, the mix­tures were pre-incu­bated for 15 min (pH 7.0) at dif­fer­ent tem­per­a­ture rang­ing from 20 to 60 °C. Resid­ual PME activ­ity was mea­sured by con­tin­u­ous spec­tro­pho­to­met­ric assay, record­ing the titra­tion of car­boxyl groups released from a pec­tin solu­tion with 0.01 M NaOH by an auto­matic pH-stat (Metr­ohm, Swit­zer­land). Rou­tine assays were per­formed with a 3.0 mg/mL cit­rus pec­tin (DE 70%, 20 ml) con­tain­ing 0.117 M NaCl [14–16]. The inhib­i­tory capac­ity was deter­mined as pec­tin­es­ter­ase-inhib­i­tory per­cent­age (%) = 100% ¡ resid­ual PME activ­ity (%) [17].

Fig. 1. SDS–PAGE anal­y­sis of PMEI expres­sion and puri­fi­ca­tion in E. coli. Lane1, pro­ tein molec­u­lar weight marker; lane 2, the puri­fied inclu­sion bodies by the nickel ion metal che­lat­ing affin­ity chro­ma­tog­ra­phy; lane 3, the unin­duced sol­u­ble frac­ tion of E. coli BL21 with pET30a-PMEI; lane 4, the induced sol­u­ble frac­tion of E. coli BL21 with pET30a-PMEI; lane 5, the unin­duced insol­u­ble frac­tion of E. coli BL21with pET30a-PMEI; lane 6, the induced insol­u­ble frac­tion of E. coli BL21 with pET30a-PMEI.



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Fig. 2. The inhib­i­tory capac­ity of recombinant PMEI against var­i­ous PMEs using gel dif­fu­sion assay. The wells were loaded with: 1, orange fruit PME; 2, orange fruit PME mixed with recombinant PMEI; 3, tomato fruit PME mixed with recombinant PMEI; 4, tomato fruit PME; 5, grape fruit PME mixed with recombinant PMEI; 6, grape fruit PME; 7, haw­thorn fruit PME mixed with recombinant PMEI; 8, haw­thorn fruit PME; 9, car­rot fruit PME; 10, car­rot fruit PME mixed with recombinant PMEI; 11, kiwi fruit PME; 12, kiwi fruit PME mixed with recombinant PMEI; 13, apple fruit PME; 14, apple fruit PME mixed with recombinant PMEI; 15, pine­ap­ple fruit PME; 16, pine­ap­ple fruit PME mixed with recombinant PMEI.

Effects of pH and tem­per­a­ture on recombinant PMEI activ­ity

Acknowl­edg­ments

The recombinant PMEI inhib­i­tory capac­ity to tomato PME was mon­i­tored at dif­fer­ent pH rang­ing from 3 to 10, the results showed that dif­fer­ent pH val­ues had no obvi­ous influ­ence on the sta­bil­ity of the PME–PMEI com­plexes. Mean­while, the recombinant PMEI inhib­i­tory capac­ity was mon­i­tored at dif­fer­ent tem­per­a­ture from 20 to 60 °C. The results dem­on­strated that recombinant PMEI effi­ ciently inhib­ited PME between 20 and 40 °C, but the inhib­i­tory capac­ity rap­idly decreased from 40 to 60 °C. The results sug­gested that recombinant PMEI could effi­ciently over­come the “cloud loss” in fruit and veg­e­ta­ble juice stor­age at room tem­per­a­ture.

We are grate­ful to Prof. Xing­hua Guo for tech­ni­cal help and dis­ cus­sion. The research was sup­ported by a National High-Tech R&D Pro­gram Grant (No. 2006AA10Z317, No. 2007AA10Z354 and No. 2006BAB04A06) from Min­is­try of Sci­ence and Tech­nol­ogy of the People’s Repub­lic of China.

Dis­cus­sion In terms of bio­log­i­cal activ­ity, the for­ma­tion of inclu­sion bodies remains a sig­nif­i­cant bar­rier in the E. coli expres­sion sys­tem, due to the ardu­ous task of refold­ing the aggre­gated pro­tein [18]. In order to decrease the for­ma­tion of inclu­sion bodies, the bac­te­rial cul­tures were grown below 37 °C. This will pro­vide suf­fi­cient time for pro­tein refold­ing since the rates of tran­scrip­tion and trans­la­ tion will sub­stan­tially decrease at lower tem­per­a­ture [19]. So, in this study, BL21 (DE3) with pET30a-PMEI was first grown at 25 °C, but the PMEI activ­ity in sol­u­ble frac­tion was extremely low (data not shown). The reason may be that PMEI con­tains five cys­te­ines res­i­due to form two disul­fide bonds, which are essen­tial for the PMEI sec­ond­ary struc­ture. Whereas the reduc­ing envi­ron­ment in E. coli cyto­plasm can not facil­i­tate exten­sive disul­fide bond for­ma­ tion [20], PMEI may not be pro­duced in their cor­rect con­for­ma­ tion in the cyto­plasm. In order to obtain a high-level pro­duc­tion of PMEI with bio­log­i­cal activ­ity, the recombinant E. coli BL21 was grown at 37 °C to form inclu­sion bodies. Then the aggre­gated pro­ tein was cor­rectly refolded in the buffer with glu­ta­thi­one redox sys­tem [21]. As a pro­kary­otic expres­sion sys­tem, E. coli can not per­form the post-trans­la­tional mod­i­fi­ca­tions that are often required for the func­tional char­ac­ter­is­tics of the eukary­otic pro­teins [9]. Although the kiwi PMEI was found to be gly­cos­yl­ated [2], the N-gly­co­si­dase F diges­tion assay showed that gly­cidic portion was bur­ied in the native pro­tein [22]. So, we attempted to choose E. coli expres­sion sys­tem to pro­duce PMEI. The results showed that N-linked gly­co­ syl­a­tion of PMEI may not be essen­tial for the func­tions of PMEI. In addi­tion, the recombinant PMEI remained sta­ble inhib­i­tor activ­ ity to tomato PME at 20–40 °C (pH 7.0). The results sug­gested that the recombinant PMEI has a potential appli­ca­tion in the fruit juice prod­ucts indus­try.

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