Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність

Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність

Нещодавно нами очищено ендогенний дефензин із проростків сосни звичайної, а також клоновано кДНК, яка кодує дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензину 1, який кодує зрілу форму цього білка, клоновано у вектор pET42а(+) та індуковано його експресію у бактеріальній систе...

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Date:2008
Main Authors: Ковальова, В.А., Гут, Р.Т., Gout, I.T.
Format: Article
Language:Ukrainian
Published: Інститут молекулярної біології і генетики НАН України 2008
Series:Біополімери і клітина
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Структура та функції біополімерів
Online Access:https://nasplib.isofts.kiev.ua/handle/123456789/157740
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Journal Title:Digital Library of Periodicals of National Academy of Sciences of Ukraine
Cite this:Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність / В.А. Ковальова, Р.Т. Гут, І.Т. Гут // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 377-384. — Бібліогр.: 26 назв. — укр., англ.

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spelling nasplib_isofts_kiev_ua-123456789-1577402025-02-23T17:41:29Z Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність Получение рекомбинантного дефензина 1 сосны обыкновенной и его антифунгальная активность Production of Scots pine recombinant defensin 1 and its antifungal activity Ковальова, В.А. Гут, Р.Т. Gout, I.T. Структура та функції біополімерів Нещодавно нами очищено ендогенний дефензин із проростків сосни звичайної, а також клоновано кДНК, яка кодує дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензину 1, який кодує зрілу форму цього білка, клоновано у вектор pET42а(+) та індуковано його експресію у бактеріальній системі Escherichia coli. Оптимізовано умови продукування рекомбінантного білка PsDef1, злитого з глутатіон-S-трансферазою, у розчинній формі. Після афінного очищення цього білка на глутатіон-сефарозі та протеолізу фактором Ха отримано біологічно активний препарат рекомбінантного PsDef1, антифунгальна активність якого співмірна з такою ендогенного дефензину 1 сосни звичайної. Recently we have purified an endogenous defensin from Scots pine germs, and cloned cDNA encoding defensin 1 (PsDef1, Pinus sylvestris defensin 1). The cDNA region encoding a mature form of Scots pine defensin 1 was cloned into a vector pET 42a(+), and the expression of recombinant GST/PsDef1 in the Escherichia coli bacterial system was induced. The conditions of production of soluble GST-proteins were optimized. After purification of the recombinant protein by affinity chromatography on Glutathione-Sepharose column and proteolytic cleavage with Factor Xa, the functionally active preparation of recombinant PsDef1 was obtained. Its antifungal activity is similar to that of endogenous Scots pine defensin 1. Ранее нами очищен эндогенный дефензин из проростков сосны обыкновенной, а также клонирована кДНК, кодирующая дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензина 1, кодирующий зрелую форму этого белка, клонирован в вектор pET42а(+) и индуцирована его экспрессия в бактериальной системе Escherichia coli. Оптимизированы условия продукции рекомбинантного белка PsDef1, слитого с глутатион-S-трансферазой, в растворимой форме. После аффинной очистки этого белка на глутатион-сефарозе и протеолиза фактором Ха получен биологически активный препарат рекомбинантного PsDef1, антифунгальная активность которого соизмерима з таковой эндогенного дефензина 1 сосны обыкновенной. 2008 Article Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність / В.А. Ковальова, Р.Т. Гут, І.Т. Гут // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 377-384. — Бібліогр.: 26 назв. — укр., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0007B5 https://nasplib.isofts.kiev.ua/handle/123456789/157740 577.112.083 uk Біополімери і клітина application/pdf application/pdf Інститут молекулярної біології і генетики НАН України
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language Ukrainian
topic Структура та функції біополімерів
Структура та функції біополімерів
spellingShingle Структура та функції біополімерів
Структура та функції біополімерів
Ковальова, В.А.
Гут, Р.Т.
Gout, I.T.
Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
Біополімери і клітина
description Нещодавно нами очищено ендогенний дефензин із проростків сосни звичайної, а також клоновано кДНК, яка кодує дефензин 1 (PsDef1, Pinus sylvestris дефензин 1). Фрагмент кДНК дефензину 1, який кодує зрілу форму цього білка, клоновано у вектор pET42а(+) та індуковано його експресію у бактеріальній системі Escherichia coli. Оптимізовано умови продукування рекомбінантного білка PsDef1, злитого з глутатіон-S-трансферазою, у розчинній формі. Після афінного очищення цього білка на глутатіон-сефарозі та протеолізу фактором Ха отримано біологічно активний препарат рекомбінантного PsDef1, антифунгальна активність якого співмірна з такою ендогенного дефензину 1 сосни звичайної.
format Article
author Ковальова, В.А.
Гут, Р.Т.
Gout, I.T.
author_facet Ковальова, В.А.
Гут, Р.Т.
Gout, I.T.
author_sort Ковальова, В.А.
title Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
title_short Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
title_full Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
title_fullStr Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
title_full_unstemmed Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
title_sort отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність
publisher Інститут молекулярної біології і генетики НАН України
publishDate 2008
topic_facet Структура та функції біополімерів
url https://nasplib.isofts.kiev.ua/handle/123456789/157740
citation_txt Отримання рекомбінантного дефензину 1 сосни звичайної та його антифунгальна активність / В.А. Ковальова, Р.Т. Гут, І.Т. Гут // Біополімери і клітина. — 2008. — Т. 24, № 5. — С. 377-384. — Бібліогр.: 26 назв. — укр., англ.
series Біополімери і клітина
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fulltext Production of recombinant Scots pine defensin 1 and characterization of its antifungal activity V. A. Kovaleva, R. T. Gout, I. T. Gout1 National University of Forestry, Henerala Chuprynky 103, Lviv, 79057, Ukraine 1University College London, Gower str., London, WCIE 6 BT, Great Britain We have recently reported affinity purification and molecular cloning of defensin (PsDef1, Pinus sylvestris defensin 1) from Scots pine germinating seeds. In this study, we describe subcloning of PsDef1 mature form into bacterial expression vector pET 42à(+) and the expression of recombinant GST/PsDef1 in Escherichia coli. The conditions for the expression and affinity purification of soluble GST/PsDef1GST were defined. Affinity purification of the recombinant GST/PsDef1 on glutathione-sepharose column and proteolytic removal of GST moiety with Factor Xa allowed us to generate functionally active preparations of recombinant PsDef1. Moreover, the antimicrobial activity of recombinant PsDef1 was found to be comparable to that of endogenous Scots pine defensin. Keywords: recombinant defensin PsDef1, expression, affinity purification, antifungal activity. In tro duc tion. Defensins are evo lu tion al ly con ser va - tive mol e cules of in nate im mu nity of plants, mol lusks, in sects and an i mals which are char ac ter ized by small size (< 10 kD), and amphipathic, b-lay ers-rich struc - ture and sta bi lized disulfide bridges [1, 2]. These se cre - tory pro teins (pep tides) are syn the sized as pre cur sors with fur ther re moval of sig nal pep tides. The ma ture form of defensins from dif fer ent spe cies has antimicrobial prop er ties [3]. Defensins are widely spread in plant king dom and could be pu ri fied from seeds, veg e ta tive and gen er a tive or gans of many an gio sperm spe cies. The genes of these de fen sive pro teins are ex pressed con sti tu tively or un - der the in flu ence of bi oti cal fac tors in the pe riph eral cell lay ers, form ing the first de fen sive line against patho gens [4, 5]. Plant defensins pos sess broad range of bi o log i cal ac tiv i ties, in clud ing antifungal [3, 5, 6], an ti bac te rial [7], in hib it ing of pro tein ases and a-amylases [8, 9] and block ing of Ñà2+- and Na+-channels [10]. Elu ci da tion of bi o log i cal prop er ties of plant defensins by in vi tro stud ies has been well com pli - mented by stud ies with the use of trans gen ic plants. Re - search from sev eral lab o ra to ries pro vided the ev i dence that overexpression of defensins re sults in the in crease of the re sis tance to patho genic in fec tions which re mains ef fec tive over sev eral gen er a tions. For ex am ples, con sti - tu tive ex pres sion of rad ish defensin RsAFP2 in to bacco in creases plant’s re sis tance against fun gal in fec tion by Alternaria longipes and A. solani, which af fects the leaves. More over, the ex pres sion of Medicago sativa defensin in the po ta toes pro vided a ro bust re sis tance against patho genic fun gus of Verti cilli um dahliae [11]. It is nec es sary to note the most stud ies on the func - tion of plant defensins have been per formed us ing the mod els of Arabidopsis thaliana and crops [12]. The mo lec u lar mech a nisms of antimicrobial re sis tance in long-lived plants, in clud ing wood spe cies, have not been elu ci dated clearly so far [13]. The re cent stud ies from our lab o ra tory have pro - vided the ev i dence that defensin from the Scots pine 377 ISSN 0233-7657. Biopolymers and cell. 2008. vol. 24. N 5. Translated from Ukrainian Ó V. A. KOVALEVA, R. T. GOUT, I. T. GOUT, 2008 seed lings has the po ten tial to bind ef fi ciently and spe cif - i cally to phosphotyrosine ma trixes. The re sults of mass spec trom e try anal y sis of pu ri fied defensin pro vided us the data re quired for the mo lec u lar clon ing of Pinus sylvestris defensin, which we termed PsDef1. A cDNA clone, cor re spond ing to PsDef1 (Acc. No. EF455616), was iso lated from a cDNA li brary of seven-days old Scots pine seed lings, which was gen er ated in our lab o ra - tory. The mo lec u lar stud ies al lowed us to de duct the amino acid se quence of defensin 1 and to carry out a com par a tive anal y sis of its pri mary struc ture and prop er - ties with defensins from var i ous plants [16, 17]. In ad di tion, we have de vel oped a new pro to col for the pu ri fi ca tion of en dog e nous defensin from Scots pine seed lings and dem on strated its antifungal prop er ties [14, 15]. To fur ther ad vance our knowl edge on bi o log i cal prop er ties of defensin 1, we have fo cused out re search on gen er at ing large quan ti ties of re com bi nant PsDef1 and pro duc ing spe cific polyclonal and monoclonal an ti - bod ies. The high level of pro tein pro duc tion can be achieved by em ploy ing prokaryotic ex pres sion sys tem, us ing GST or His-tag fu sions and Esch e richia coli. Here, we de scribe for the first time con struc tion of an ex press - ing plasmid cod ing for GST/ PsDef1 fu sion pro tein, af - fin ity pu ri fi ca tion of re com bi nant pro tein and the re - moval of GST moi ety by proteolytic cleav age with Fac - tor X. The re sult ing prep a ra tion of ma ture form of PsDef1 was shown to pos sess an ti bac te rial prop er ties which are com pa ra ble to those of en dog e nous defensin pu ri fied from Scots pine seed lings. Ma te ri als and meth ods. The ex pres sion vec tor pET42a(+) (‘Novagen’, USA) and E. coli strains XL-1 Blue and BL21 (DE3) have been used for clon ing and ex pres sion stud ies. The phytopathogenic fun gus cul - tures of Fusarium oxysporum YKM F-52897, F. solani YKM F-50639, Bo try tis cinerea YKM F-16753, Altermaria al ter nate YKM F-16752 and phytopathogenic bac te ria of Erwinia carotovora YKM B-1075 have been kindly pro vided by D.K. Zabolotny In sti tute of Mi cro bi ol ogy and Vi rol ogy, Na tional Acad emy of Sci ences of Ukraine. The necrotrophic fun gus cul ture of Heterobasidion annosum has been kindly pro vided by Dr. V. Kramarec (Lviv Na tional For estry Uni ver sity of Ukraine). The cul ture of oomicete Pythium dimorphum has been ob tained from the In sti tute of Forestry Research (IBL, Poland). Clon ing of ma ture form of Scots pine defensin into pET42a(+) vec tor. A frag ment of cDNA cor re spond - ing to ma ture form (ba sic do main) of Scots pine defensin 1 (mPsDef1) was am pli fied by poly mer ase chain re ac tion (PCR) us ing a tem plate cDNA clone for defensin 1 and two prim ers: di rect primer CR765 (5’-CCATTCCATGGGAATGTGCAAAACCCCCA G-3’) which con tains nu cle o tide se quences cor re - spond ing to re stric tion endonuclease NcoI, fol lowed by ini ti at ing Met and Gly in stead of Arg at the N-ter mi - nus of ma ture form of Scots pine defensin 1; re verse primer CR764 (5’-CATGAGAATTCTCAAGGGCA GGGTTTGTA-3’) in cludes nu cle o tide se quences for re stric tion endonuclease EcoRI, stop-codon and 3’- cod ing frag ment of defensin 1. The con di tions for PCR am pli fi ca tion have been de scribed pre vi ously [17]. PCR prod ucts were an a - lyzed by 1.5% agarose gel elec tro pho re sis in Tris-bo - rate buffer, pH 8.3 (50 mM Tris-H3BO3, 2 mM EDTA) un der 20 V/sm2. Am pli fied prod ucts were eluated from the gel with the use of a gel ex trac tion kit from Qiagen (USA). Pu ri fied DNA and pET42a(+) vec tor have been hy dro lyzed with restrictases NcoI/EcoRI and then pre - cip i tated us ing a stan dard method [18]. The T4 DNA-ligase (‘Fermentas’, Lith u a nia) was used for link ing am pli fied cDNA frag ment of defensin 1 into pET42a(+) vec tor in frame with the N-ter mi nally lo - cated GST. The prod ucts of li ga tion were trans formed in to XL-1 Blue com pe tent cells. The plasmid pu ri fi ca - tion kit was used to pu rify plasmid DNA from an ti bi - otic re sis tant col o nies. The pres ence of PsDef1 cDNA in sert in pu ri fied plasmids was de tected by re stric tion anal y sis with endonucleases XhoI and EcoRI as well as by PCR anal y sis with prim ers of C765 and CR764. The se quence of am pli fied PsDef1 in three re sult ing plasmids was verified by sequence analysis on automatic DNA sequencer ABI 73 TM. The ex pres sion of re com bi nant PsDef1 in bac te rial sys tem E. coli. BL21(DE3) com pe tent cells were trans - formed with pET42a/mPsDef1 con struct and ob tained col o nies were grown at 37oC in LB-me dium con tain ing 50 mg /ml kanamycin. When the op ti cal den sity of bac - te rial cul tures reached A600 = 0.6, 0.4 mM isopropyl-thiogalactosid (IPTG) was added to in duce the ex pres sion of re com bi nant pro tein. Af ter the in cu - ba tion of in duced cul tures for 2 hours, bac te rial cells 378 KOVALEVA V. A., GOUT R. T., GOUT I. T. were col lected by centrifugation at 4000 g for 20 min - utes. The pel let of bac te ria was washed with ice-cold PBS, frozen and preserved at -70 Ñ. The pu ri fi ca tion of re com bi nant PsDef1 con ju - gated with glutathion-S-transferase by af fin ity chro - ma tog ra phy. All pu ri fi ca tion steps were per formed at 4 Ñ. Cell pel let ( 2.5 g) was re sus pend and lysed in 15 ml buffer A, con sist ing of: 10 mM Tris-HCl, (pH 7.5), 150 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Tri ton X-100, 1mM PMSF with proteinase cock tail of in hib i - tors (‘Roche’, France). Cells were de stroyed by me - chan i cal grind ing in a ho mog e nizer. The lysate of cells was cen tri fuged at 17000 g for 20 min. The supernatant was in cu bated on the wheel for 2 hours with a 50% sus - pen sion of Glutathione-sepharose (‘Amersham’, Great Brit ain), pre-washed in buffer A. The beads were washed on the wheel with 50mM Tris-HCl, (pH 7.5), 150 mM NaCl with 0.05% of Twin-20 three times for 5 min utes and twice with 50 mM Tris-HCl, (pH 8.0). Bound pro teins were eluated from Glutathione-sepharose with 50 mM re duced glutathione in 50 mM Tris-HCl, (pH 8.0), 150 mM NaCl. The eluted frac tions were ana lysed by Brad ford as say to mea sure pro tein con cen tra tions and by SDS-PAGE for ex am in ing the qual ity of pu ri fied GST-PsDef1. The frac tions, which con tained GST-PsDef1 were com bined and di a lyzed against buffer B, con tain ing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, in or der to re move glutathione. The con cen tra tion of gen er ated prep a ra tions of GST-PsDef1 was mea sured by Brad ford as say. Pro - duced GST-PsDef1 was stored at -20 Ñ in presence of 50% glycerol. Pro duc tion of ma ture form of PsDef1. The Fac tor Xa (‘Sigma’, USA) was used to cleave re com bi nant Scots pine defensin (PsDef1) from the fu sion polypeptide GST-PsDef1. In brief, GST-PsDef1 was sorbed on the glutathione-sepharose and equil i brated with buffer con tain ing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2mM CaCl2. Then, Fac tor Xa was added at the con cen tra tion of 2 mg per 100 mi cro grams of GST-PsDef1. The re ac tion was per formed un der room tem per a ture for 5 hours on the wheel. Beads were then col lected by centrifugation at 3000 g for 1 min ute. The supernatant liq uid was col lected and loaded onto Centricon YM 30 (‘Millipore’, USA) for con cen tra tion at 10000 g. The fil trate was col lected and pro tein con - cen tra tion mea sured by Brad ford as say and an a lyzed by SDS-elec tro pho re sis in 15% PAAG in a Tris-tricine buffer system [19]. Antimicrobial ac tiv ity of Scots pine re com bi nant defensin 1 as say. To study the ac tiv ity of gen er ate prep a - ra tions of re com bi nant defensin 1 the pieces of fun gus my ce lium were placed into the cen ter of Petri dishes (90x15 mm) with 1.8% po tato-dex trose agar. When the di am e ter of fun gus col ony had reached 3 cm the ster ile discs of fil ter pa per were placed onto the agar 0.5 cm away from the col ony edge. The aliquots (100 ml) of dif - fer ent pro tein con cen tra tions of PsDef1 were ap plied on the pa per discs. Dis tilled wa ter has been used as a neg a - tive con trol and dif fer ent con cen tra tions of en dog e nous defensin pu ri fied from Scots pine seed lings were also tested. The plates were then in cu bated at 23 °Ñ un til my - ce lium en vel oped the disks with con trol so lu tion, while growth-in hib it ing zones were formed around the discs con tain ing recombinant defensin 1. A quan ti ta tive anal y sis of the antifungal ac tiv ity of Scots pine defensin was per formed as pre vi ously de - scribed [20]. The spores were ex tracted from sporulating fun gus cul tures, which have been grown on the 1.8%-po tato-dextose agar. The spore sus pen sion has been fil tered with dou ble layer of ster ile gauze. 80 microlitres of spore sus pen sion (2x104 spores/ml) in po tato-dex trose broth and 20 microlitres of ster ile defensin so lu tion (with con cen tra tions 0.2, 0.5, 1, 5,10 mg/ml) were added into the wells of 96-well microtiter plate. The plates were in cu bated in dark at 23 Ñ. The test sam ple con sisted of 20 milli litres of ster ile distillated wa ter. Af ter 48 hours of in cu ba tion, the op ti - cal den sity of fun gal sus pen sion was measured in each well under 595 nm. Re sults and dis cus sion. The pres ence of an endoplasmic re tic u lum sig nal pep tide, which is re - moved dur ing the pro tein pro cess ing, is a fea ture of all plant defensins [3]. The se quence anal y sis of PsDef1 cDNA clones in di cated an open read ing frame of 83 amino acid res i dues. The first 33aa cor re spond to the N-ter mi nal sig nal pep tide and re main ing 50aa form a ba sic do main of ma ture defensin. To pro duce large quan ti ties of re com bi nant ma ture form of defensin 1 we de cided to em ploy bac te rial ex pres sion sys tem of E. coli, as it al lows ef fi cient, fast and rel a tively in ex pen - 379 PRODUCTION OF RECOMBINANT SCOTS PINE DEFENSIN 1 sive way of gen er at ing re com bi nant pro tein. Since the eukaryotic sig nal se quence is not rec og nized in the prokaryotic sys tem, we have cloned a DNA frag ment, coding the mature form of PsDef1 into bacterial expression vector. A DNA frag ment, cor re spond ing to ma ture form of mPsDef1, was am pli fied by PCR us ing PsDef1 cDNA as a tem plate and two spe cific prim ers. Agarose gel elec tro pho retic anal y sis of PCR prod ucts re vealed a ma jor band of ap prox i mately 200bp which closely cor - re lates to ex pected size of 174 bp. (Fig ure 1, a). The prod uct of am pli fi ca tion was di gested with NcoI and XhoI and li gated into linearized pET42a(+) vec tor. The XL-1 Blue com pe tent cells were trans formed with the li ga tion mix and plated for an ti bi otic se lec tion. Plasmid DNA was pu ri fied from col o nies grown on agar plates con tain ing kanamycin. The pres ence of PsDef1 in sert in pu ri fied plasmid DNAs was ana lysed by PCR am pli - fi ca tion (Fig ure 1b) and re stric tion anal y sis with NcoI and XhoI endonuclease (Fig ure 1c). The ob tained re - sults clearly in di cate that PsDef1 cDNA is present in 4 out of 6 plasmid preparations. To study ex pres sion the BL21 (DE3) com pe tent cells were trans formed by the gen er ated pET42a/mPsDef1 plasmid . In par al lel, the vec tor (pET42a alone) was also in tro duced into the BL21 (DE3) com pe tent cells. The ex pres sion of re com bi nant GST-PsDef1 was in duced in BL21 (DE3) by the ad di - tion of 1mM IPTG. The SDS-PAGE anal y sis of to tal lysates from in ducted cells re vealed the pres ence of re - com bi nant pro tein of an ex pected mo lec u lar weight, 35.5 kD (Fig ure 2). This anal y sis also showed that GST-PsDef1 fu sion pro tein is sol u ble in buffer con - tain ing 1%Tri ton X-100. We also found that to tal cell lysate con tained ap prox i mately 15% of GST-PsDef1 fu sion pro teins. No ta bly, some amounts of GST-PsDef1 were also observed among detergent-insoluble proteins. Tak ing into ac count that re com bi nant GST-PsDef1 was found in both Tri ton sol u ble and in sol u ble frac - tions, in the form of in clu sion bod ies, we fo cused our ef forts on op ti miz ing the con di tions for gen er at ing cul - tur ing max i mum level of tri ton-sol u ble GST-PsDef1. By test ing var i ous tem per a tures for the ex pres sion of GST-PsDef1 we found that the op ti mal tem per a ture for 380 KOVALEVA V. A., GOUT R. T., GOUT I. T. Fig.1. Cloning of a cDNA fragment encoding mature form of the Scots pine defensin 1: (a) 1 – the products of PCR amplification of cDNA PsDef1; Ì – 1 kb Plus DNA Ladder GibñoBRL; (b) the analysis of pET42a/mPsDef1 plasmid by PCR (lane 1); Ì – 1 kb DNA Ladder Fermentas; (c) restriction analysis of pET42a/mPsDef1 plasmid using endonucleases Xho 1 and Nco 1 (lane 1); Ì – 1 kb DNA Ladder Fermentas. Fig.2. SDS-PAAG results of GST-PsDef1 exspession in E. coli BL21(DE3) strain: 1, 2, 3, 4 – fractions of the total cell lysates; 5, 6, 7, 8 – triton-soluble protein fractions; Ì – broad-range protein standards BioRad. Lanes 1, 2, 5, 6 –uninduced bacterial cultures; 3, 4, 7, 8 – proteins from the cells induced by the addition of IPTG; 1, 3, 5, 7 – the cells transformed by plasmid without insert; 2, 4, 6, 8 –the cells, transformed by recombinant vector. Amount of proteins on each of the lines is equivalent to the 100 µl cell suspension. gen er at ing the high est level of re com bi nant GST-PsDef1 is 37 °C, while low er ing the in duc tion tem per a ture to 28-30°C(of ten used in or der to fold polypeptide chain of re com bi nant prod uct cor rectly) re sulted in no tice able re duc tion of tri ton-sol u ble re - com bi nant pro tein. The same ef fect was no ticed with the in crease of the in duc tion time. In sum mary, the op - ti mal ex pres sion of sol u ble GST-PsDef1 was achieved by in cu bat ing trans formed cells with 0.4mM IPTG for 2 hours at 37°Ñ. The use of pET42a ex pres sion plasmid im plied the pro duc tion of re com bi nant PsDef1, con ju gated with GST. The ex pres sion of PsDef1 fused to GST al lowed us to em ploy af fin ity chro ma tog ra phy on glutathione-sepharose. As shown in Fig ure 3a, af fin ity pu ri fi ca tion from cells ex press ing plasmid alone or pET42a/mPsDef1 re sults in highly en riched prep a ra - tions of GST and GST-PsDef1 re spec tively. Both prep - a ra tions con tain some mi nor bands which ex hibit sim i - lar pat tern in both sam ples. In or der to elim i nate the pos si bil ity of non-spe cific pro tein sorbtion onto glutathione-sepharose, we in cu bated af fin ity beads with Tri ton-sol u ble pro teins of uninduced cells. The re - sults pre sented in Fig ure 3b (lane 3), show very lit tle of non-spe cific bind ing to Glutatione sepharose from lysates of uninduced cells. We have also elim i nated the pos si bil ity that ob served mi nor bands are bind ing part - ners of re com bi nant GST and GST-PsDef1, as they were not eluted from the af fin ity ma trixes in the pres - ence of 0.5 M NaCl, but were readily eluated with 50 mM of re duced glutathione. As seen in the spec trum of tri ton-sol u ble pro teins (Fig ure 3b, lanes 1 and 2) mi nor bands ap pear only af ter the in duc tion of re com bi nant pro teins. There fore, the mi nor pro teins are the prod ucts of ei ther deg ra da tion or in com plete trans la tion of GST-PsDef1. The affinity purified preparations of recombinant GST-PsDef1 contained approximately 75% the fusion protein. The GST-PsDef1 fu sion pro tein has mo lec u lar mass of ap prox i mately 35.5 kDa. The fu sion con sists of 220 amino ac ids of GST at the N-ter mi nus and 51aa cor re spond ing to ma ture form of PsDef1. There is also a 58aa in sert be tween GST and PsDef1, which con tains Fac tor X re stric tion site and some se quences from pET42a vec tor. Fac tor Xa is a serine proteinase, which rec og nizes and hydrolyzes the se quences of Ile Glu Gly Arg af ter cer tain amino acid se quences. The clon ing strat egy of pET42a/PsDef1 ex pres sion vec tor al lows proteolytic re moval of the ma ture form of PsDef1, con - sist ing of 52 amino-ac ids, where the first two amino ac - ids are the ini ti at ing methionine and then glycine instead of arginine. To re move GST moi ety from the GST-PsDef1fusion pro tein, we tested two cleav age pro to cols. Vari ant A: the cleav age of re com bi nant GST-PsDef1with Fac tor Xa was car ried out in so lu tion. 381 PRODUCTION OF RECOMBINANT SCOTS PINE DEFENSIN 1 Fig.3. Isolation of the recombinant Scots pine defensin 1 from the E. coli BL21(DE3) cell lysates: (à) purification of recombinant GST?PsDef1 onto affinity sorbent: 1, 2 – triton-soluble proteins from the cells transformed by pET-42à and pET-42à-ÐsDef1 plasmids, respectively; 3 – GST preparation; 4 – GST-ÐsDef1 preparation; (b) 1, 2 – triton-soluble proteins from the uninduced and induced cells transformed by pET-42à/mDef1 plasmid, respectively; 3, 4 – proteins that bound to glutathione-Sepharose from lysates of the uninduced and IPTG-induced cells, respectively. Proteins were separated by 12% SDS-PAAG electrophoresis in Laemmle’s system; (c) ? fractions after the proteolytic digestion of GST- ÐsDef1 with Õà factor: 1, 2 – immobilized proteins on glutathione-Sepharose before and after digestion, respectively. 15% Tricine-SDS-PAAG was performed for separation of each fraction. Gels were stained by Coomassie G-250. The re moval of cleaved GST was per formed on Glutathione-sepharose. The sep a ra tion of Fac tor Xa from cleaved PsDef1was car ried out by centrifugation on Centricon YM 30 col umns (‘Millipore’). Vari ant B: pro te ol y sis of the GST-PsDef1 fu sion pro tein was per formed on Glutathione sepharose. Here, Fac tor Xa was added to the Glutathione-sepharose sus - pen sion with im mo bi lized re com bi nant GST-PsDef1. Af ter the cleav age, PsDef1 was re leased into so lu tion, which was then pro cessed for the re moval of Fac tor Xa proteinase as de scribed above. Hav ing both pro to cols com pared, we found that the cleav age on beads is faster, more eco nom i cal and al lows greater output of PsDef1 protein. Fur ther stud ies in di cated that op ti mal cleav age of GST-PsDef1 re quires 20mkg of Fac tor Xa per 1 mg of fu sion pro tein. The time-course anal y sis of Fac tor Xa cleav age showed that full cleav age of GST-PsDef1 re - quires 5 hours at room tem per a ture (Fig ure 3, c). As seen in lane 2, the band cor re spond ing to the GST-PsDef1fusion dis ap pear af ter 5 hrs of di ges tion, while the ma jor band of ap prox i mately 30kDa, which cor re spond to GST alone, is clearly detected. So, the pro to col de signed for Fac tor Xacleav age of GST-PsDef1 al lows the pro duc tion of ho mo ge neous prep a ra tions of re com bi nant PsDef1. The fi nal out put of PsDef1was ap prox i mately 10% from to tal GST/PsDef1 fu sion pro tein, im mo bi lized on Glutathione-sepharose. The ac tiv ity of re com bi nant pro teins de pends on cor rect fold ing of their polypeptide chain. Ter tiary struc ture of plant defensin con sists of three antiparallel b-sheets and an a-he lix, sta bi lized with 4 disulfide bridges. So, the ac tiv ity of re com bi nant defensin could be lost from in ad e quate for ma tion of -Cys-Cys pairs. The study of mech a nisms of ac tion of plant defensins, per formed in the Braeckert group [23], re vealed sig nif - i cant role of two re gions in Rs-AFP2 which me di ated the in ter ac tion with re cep tors on the fun gus mem brane. These re gions are formed by amino acid res i dues, lo - cated in dif fer ent parts of the polypeptide chain. For ex - am ple, one re gion in volves Thr10, Ser12, Leu28 and Phe 49. The site-di rected sub sti tu tion of these amino ac ids with amino res i dues which disturbe the 3D struc - ture of defensin, leads to the loss of antifungal activity. Next, we tested the antifungal ac tiv ity of re com bi - nant GST-PsDef1 and cleaved PsDef1. In this study, we used the fungi from the ge nus of Fusarium. No antifungal ac tiv ity was ob served in the pres ence of re - com bi nant GST/PsDef1 (even, when the con cen tra tion 382 KOVALEVA V. A., GOUT R. T., GOUT I. T. Fig.4. Inhibitory effect of purified recombinant ÐsDef1 on the growth of Heterobasidion annosum (à) and Fusarium solani (b): K - the sterile distilled water; 1 – 5 µg of preparation; 2 – 10 µg of preparation. of GST/PsDef1 in the cul ture me dium was 50 mg/ml). When, the ac tiv ity of PsDef1 was tested in the growth in hib it ing as say, we reproducibly ob served strong antifungal prop er ties of gen er ated prep a ra tions of PsDef1. The antifungal ac tiv ity was mea sured by the for ma tion of zones, where the growth of my ce lium of H. annosum and F solani was inhibited (picture 4). These find ings pro vided the vi sual ev i dence for the in hi bi tion of fun gal growth by re com bi nant PsDef1. The next task was to de velop the as say which would al - low the quantitation of PsDef1 antifungal ac tiv ity. To do so, we em ployed the microspectrophotometric method in which the op ti cal den sity of fungi spore sus - pen sion was mea sured af ter 48 hours of in cu ba tion in po tato-dex trose broth, con tain ing var i ous con cen tra - tions of re com bi nant defensin. When max i mum con - cen tra tion (4 mi cro grams/mil li li ter) of PsDef1 was used, the in hi bi tion of fun gal growth was: for F solani by 81%, for F oxysporum by 50%, for H annosum by 71%, for Pythium dimorphum by 84%. These re sults in di cate dif fer en tial ac tiv ity of re com bi nant PsDef1 to - wards dif fer ent fungi. Dif fer en tial in hib i tory ef fect of plant defensins to wards dif fer ent fungi spe cies has been pre vi ously de scribed [24]. The ac tiv ity of antimicrobial prep a ra tions is mea - sured by the value of IC50. We have es ti mated IC50 of re - com bi nant PsDef1 for F solani, F oxysporum, B. cinerea, and P. dimorphum. Their rates are 1.4, 4.0, 2.6, 1.2, 2.0 mi cro grams per mil li li ter re spec tively. The defensin PgD1 from Picea glauca, which ex hib its 80% iden tity to PsDef1, has been re cently ex pressed in bac - te rial sys tem and its antimicrobial ac tiv ity has been stud ied. The pu ri fied re com bi nant prep a ra tions of PgD1 in hib ited the growth of F. oxysporum by 95.2%, which is sim i lar to that of Scots pine defensin 1 [25]. In pre vi ous stud ies, we re searched the antifungal ac tiv ity of en dog e nous defensin pu ri fied from Scots pine seed lings [15, 26]. These re sults al lowed us to com pare the val ues of IC50 for re com bi nant and en dog - e nous defensin. The re sults of this com par a tive anal y - sis are rep re sented in the di a gram of Fig ure 5. We found that the ac tiv ity of re com bi nant PsDef1 is 1.5-2 times lower then that of en dog e nous defensin from seed lings. This is prob a bly the re sult of par tial pro te ol - y sis of PsDef1 at Gly-Arg44 site, which might be re - cog nised by Fac tor Xa. In sum mary, we de scribed for the first time the ex - pres sion and af fin ity pu ri fi ca tion of re com bi nant defensin 1 from Scots pine pos sess ing a strong antifungal ac tiv ity. The avail abil ity of re com bi nant PsDef1 pro vides us with an ex cel lent op por tu nity to study bio chem i cal and func tional prop er ties of plant defensins and to elu ci date mo lec u lar mech a nisms of their ac tion. In ad di tion, re com bi nant prep a ra tion of PsDef1 will be used for mak ing spe cific polyclonal and monoclonal an ti bod ies and to study the ex pres sion pro - file of defensin 1 in re sponse to var i ous growth-regulatory agents and pathogenic organisms. Â. À. Êî âàëü î âà, Ð. Ò. Ãóò, ². Ò. Ãóò Îòðè ìàí íÿ ðå êîìá³íà íòíî ãî äå ôåí çè íó 1 ñî ñíè çâè ÷àé íî¿ òà éîãî àí òè ôóí ãàëü íà àê òèâí³ñòü Ðå çþ ìå Íå ùî äàâ íî íàìè î÷è ùå íî åí äî ãåí íèé äå ôåí çèí ³ç ïðî ðîñòê³â ñî ñíè çâè ÷àé íî¿, à òà êîæ êëî íî âà íî êÄÍÊ, ÿêà êîäóº äå ôåí çèí 1 (PsDef1, Pinus sylvestris äå ôåí çèí 1). Ôðàã ìåíò êÄÍÊ äå ôåí - çè íó 1, ÿêèé êîäóº çð³ëó ôîð ìó öüî ãî á³ëêà, êëî íî âà íî ó âåê òîð pET42à(+) òà ³íäó êî âà íî éîãî åêñïðåñ³þ ó áàê òåð³àëüí³é ñèñ - òåì³ Escherichia coli. Îïòèì³çî âà íî óìî âè ïðî äó êó âàí íÿ ðå - êîìá³íà íòíî ãî á³ëêà PsDef1, çëè òî ãî ç ãëó òàò³îí-S-òðàíñ ôå ðà çîþ, ó ðîç ÷èíí³é ôîðì³. ϳñëÿ àô³ííî - ãî î÷è ùåí íÿ öüî ãî á³ëêà íà ãëó òàò³îí-ñå ôà ðîç³ òà ïðî òå îë³çó ôàê òî ðîì Õà îò ðè ìà íî á³îëîã³÷íî àê òèâ íèé ïðå ïà ðàò ðå - êîìá³íà íòíî ãî PsDef1, àí òè ôóí ãàëü íà àê òèâí³ñòü ÿêî ãî ñï³âì³ðíà ç òà êîþ åí äî ãåí íî ãî äå ôåí çè íó 1 ñî ñíè çâè ÷àé íî¿. Êëþ ÷îâ³ ñëî âà: ðå êîìá³íà íòíèé äå ôåí çèí PsDef1, åêñïðåñ³ÿ, àô³ííå î÷è ùåí íÿ, àí òè ôóí ãàëü íà àê òèâí³ñòü. 383 PRODUCTION OF RECOMBINANT SCOTS PINE DEFENSIN 1 Fig.5. Comparative analysis of the antifungal activity of the endogenous PsDef1(1) with that of its recombinant analog (2). Â. À. Êî âà ëå âà, Ð. Ò. Ãóò, È. Ò. Ãóò Ïî ëó ÷å íèå ðå êîì áè íàí òíî ãî äå ôåí çè íà 1 ñî ñíû îá ûê íî âåí íîé è åãî àí òè ôóí ãàëü íàÿ àê òèâ íîñòü Ðå çþ ìå Ðà íåå íàìè î÷è ùåí ýí äî ãåí íûé äå ôåí çèí èç ïðî ðîñ òêîâ ñî ñíû îá ûê íî âåí íîé, à òàê æå êëî íè ðî âà íà êÄÍÊ, êî äè ðó þ ùàÿ äå ôåí - çèí 1 (PsDef1, Pinus sylvestris äå ôåí çèí 1). Ôðàã ìåíò êÄÍÊ äå - ôåí çè íà 1, êî äè ðó þ ùèé çðå ëóþ ôîð ìó ýòî ãî áåë êà, êëî íè ðî âàí â âåê òîð pET42à(+) è èí äó öè ðî âà íà åãî ýêñ ïðåñ ñèÿ â áàê òå ðè àëü - íîé ñèñ òå ìå Escherichia coli. Îïòè ìè çè ðî âà íû óñëî âèÿ ïðî äóê - öèè ðå êîì áè íàí òíî ãî áåë êà PsDef1, ñëè òî ãî ñ ãëó òà òè îí-S-òðàíñ ôå ðà çîé, â ðàñ òâî ðè ìîé ôîð ìå. 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