Research Article Highly Effective Renaturation of a ...downloads.hindawi.com/journals/bmri/2014/324705.pdf · Highly Effective Renaturation of a Streptokinase from Streptococcus pyogenes

Research ArticleHighly Effective Renaturation of a Streptokinase fromStreptococcus pyogenes DT7 as Inclusion Bodies Overexpressedin Escherichia coli

Sy Le Thanh Nguyen1 Dinh Thi Quyen12 and Hong Diep Vu1

1 Institute of Biotechnology Vietnam Academy of Science and Technology 18 Hoang Quoc Viet Road District of Cau GiayHanoi 10600 Vietnam

2Department of Biotechnology and Pharmacology University of Science and Technology of Hanoi 18 Hoang Quoc Viet RoadDistrict of Cau Giay Hanoi 10600 Vietnam

Correspondence should be addressed to DinhThi Quyen quyenibtacvn

Received 12 November 2013 Revised 28 February 2014 Accepted 31 March 2014 Published 5 May 2014

Academic Editor Noomen Hmidet

Copyright copy 2014 Sy LeThanh Nguyen et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering fromcardiovascular diseases We reported highly effective renaturation of a SK from S pyogeness DT7 overexpressed in E colipurification and biochemical characterization A gene coding for the SK was cloned from S pyogenessDT7 Because accumulationof active SK is toxic to the host cells we have expressed it in the form of inclusion bodiesThemature protein was overexpressed in Ecoli BL21 DE3pESK under the control of the strong promoter tac induced by IPTGwith a level of 60 of the total cell proteinsTheactivity of the rSK renatured in phosphate buffer supplementedwith Triton X-100 and glycerol was covered with up to 41 folds of itsinitial activity The purified of protein was identified with MALDI-TOFmass spectrometry through four peptide fragments whichshowed 100 identification to the corresponding peptides of the putative SK from GenBank Due to overexpression and highlyeffective renaturation of large amounts of inclusion bodies the recombinant E coli BL21 DE3pESK system could be potentiallyapplied for large-scale production of SK used in the therapy of acute myocardial infarction

1 Introduction

Streptokinase (EC 349922) (SK) a commercially importantnonprotease binds stoichiometrically to both circulatingand thrombus-bound plasminogen (Plg) to generate SK-plasminogen activator complex Cleavage of plasminogen inzymogen form at an Arg-Val bond generates plasmin anactive enzyme that degrades fibrin component of thrombin[1] Due to this property the streptokinase has been widelyused in the therapy of acute myocardial infarction for itsstrong activity in dissolving blood clots [2]

Most groupA C andG120573-hemolytic streptococci isolatedfrom human hosts secrete streptokinase with molecular massof 47 kDa which convert the plasminogen to the serineprotease plasmin However due to low SK production yieldsfrom natural host and its pathogenicity so research interest

has shifted to cloning and expression of SK in hyperproduc-tive and safe heterologous host systems Therefore sk geneshave been cloned and expressed in different expression sys-tems including Bacillus subtilis [3] Streptococcus sanguis [4]Streptomyces lividans [5 6] Schizosaccharomyces pombe [7]Pichia pastoris [1 8] Lactococcus lactis [9] and Escherichiacoli [10 11] However there are some disadvantages ofproducing recombinant proteins inPichia pastorisdue to highglycosylation level [12] or in Lactococcus lactis due to low celldensity [9]

Escherichia coli is the most commonly used host forthe production of recombinant proteins both in researchand industry [13] High-level expression of recombinantproteins in the form of a soluble intracellular productsecretory product or as insoluble inclusion bodies dependson promoter system host-vector interactions sequence and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 324705 9 pageshttpdxdoiorg1011552014324705

2 BioMed Research International

characteristics of recombinant products and the effect of theexpressed foreign protein on host cell physiology [14]

The expression of SK as inclusion bodies byE coli systemsis shown to be useful for obtaining large amounts of proteinprovided that renaturation is effective and recovery of activeprotein is high Thus the purpose of this study was firstlyto overproduce the recombinant streptokinase in E coliBL21 (DE3) and simultaneously to refold effectively the largeamount of the recombinant streptokinase as inclusion bodiesoverexpressed by E coli BL21 (DE3) under the control ofthe promoter T7 Only both objectives were gained then therecombinant E coli overproducing SK as inclusion bodiescan become a potential strain for industrial SK produc-tion

2 Materials and Methods

21 Chemicals and Reagents DNA cloning kit RNase Arestriction enzymes (BamHI NotI and EcoRI) T4-ligaseand Proteinase K were purchased from Fermentas (ThermoFisher Scientific Inc Waltham USA) The DNA ExtractionKit was from Qiagen (Venlo Netherlands) Protein Extrac-tion Kit and ProBond resin were supplied by InvitrogenCorp (Carlsbad CA USA) Human plasminogen from MPBiomedicals (Santa Ana USA) SK N (p-tosyl) gly-pro-lys-4-nitro anilide acetate salt (AAS) SDS from Sigma Aldrich Co(St Luis USA) Plasminogen Tween 20 and Tween 80 fromBioBasic Inc (NYUSA) TritonX-100 andEDTA fromMerck(Darmstadt Germany) All other reagents were of analyticalgrade unless otherwise stated

22 Plasmids Bacterial Strains and Culture Conditions Thebacterial strain Streptococcus pyogenes DT7 (GQ247718) iso-lated from a patient at the Army Hospital No 103 (HanoiVietnam) was used as the source of the streptokinase (sk)gene Escherichia coli DH5120572 (Fminus oslash80dlacZΔM15 Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17(rKminus mK+) phoAsupE44 120582ndash thi-1 gyrA96 relA1) and the vector pJET12blunt(Fermentas Thermo Fisher Scientific Inc Waltham USA)were used for DNA manipulations and amplificationEscherichia coli BL21 (DE3) cells (FndashompT gal dcm lon hsdSB(119903119861

minus119898119861

minus) 120582(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])and pET22b+ vector (Novagen Merck KGaA DarmstadtGermany)were used for expression of SK LBmedium (Luria-Bertani) containing 1 (wv) bacto tryptone 05 (wv) yeastextract 1 (wv) NaCl pH 7ndash75 was used for cultivation ofE coliDH5120572 and BL21 (DE3) LB agar contained additionally2 (wv) agar and 100 120583g ampicillinmL

23 DNA Manipulations Genomic and plasmid DNA isola-tion was carried out by methods which have been previouslydescribed [15] DNA fragments and PCR products wereexcised from a 08 agarose gel and purified by a gelextraction kit (Qiagen Venlo The Netherlands) accordingto the manufacturerrsquos instructions DNA sequencing wasperformed on an ABI PRISM 3100 Avant Genetic Analyzer(Applied Biosystems Inc Foster City USA) E coli DH5120572

and BL21 were transformed using heat shockmethod that hasbeen previously described [15]

24 DNA Amplification and Plasmid Construction The puta-tive sk-coding DNA fragment was amplified from S pyogenesDT7 genomic DNA by PCR with Taq DNA polymeraseBased on the nucleotide sequence of the sk gene fromS pyogenes strain (GenBank Z48617) 3 oligonucleotidesmSKF GGC GGATCC CATATG ATTGCTGGACCTG andSKF GCC CAT GGG CAA AAA TTA CTT AT and SKRGCC TCG AGT TTG TCB TTA GGG TT were designed asprimers for introduction of the underlined BamHI and XhoIrestriction sites respectively The PCR mixture contained25 120583L 10x PCR buffer 2 120583L of 25mMdNTP 25 120583L of 25mMMgCl

2

05 120583L genomic DNA (50ndash100 ng) 025120583L 5 unit Taqpolymerase and 1 120583L each primer (10 pmol) supplementedwith 1525120583L distillated water to a final volume of 25120583L Thethermocycler conditions were as follows 95∘C41015840 30 cycles of(95∘C3010158401015840 52∘C4510158401015840 72∘C4510158401015840) 72∘C101015840The PCR productsamplified from the genomic DNA with the primer pair SKFand SKR were inserted into the cloning vector pJET12bluntresulting in pJSK DNA sequencing was performed on ABIPRISM 3100 Avant Genetic Analyzer Sequence alignmentswere constructed and analyzed using the programMegAlignDNAStar It was followed by ligation of the BamHI-XhoIdigested PCR products (with the primer pairmSKF and SKR)with pET22b+ linearized by the same enzymes resultingin pESK under the control of the T7-promoter induced byisopropyl-120573-D-thiogalactopyranoside (IPTG) and possessingthe ampicillin marker The streptokinase rSKhis encodedby the plasmid pESK contains the mature streptokinasefused with the 6x histidine-tag and no native leader seq-uence

25 rSK Expression The transformant E coli BL21pESK wascultivated overnight in 5mL of LB medium containing 5120583Lof 100mgmL ampicillin at 37∘C on an orbital shaker at200 rpm Overnight culture (2mL) was inoculated in a 1-liter Erlenmeyer flask containing 200mL of LB broth and200120583L of 100mgmL ampicillin The culture was grownat 37∘C with agitation at 200 rpm and until an opticaldensity (OD) at 600 nm reached 06 (for approximately 25 h)then 200 120583L of 100mM IPTG was added The culture wascontinuously incubated at 37∘C with agitation at 200 rpm for3ndash6 h induction Cells were harvested by centrifugation at6000 rpm for 10min at 4∘C Wet weight cells were used forprotein purification

26 Purification of Streptokinase The fusion form rSKhis car-rying a C-terminal 6xHis tag was expressed in E coli BL21 Topurify rSK 100mg wet weight cells from a 120mL culture inLB medium were harvested by centrifugation and suspendedin 10mL of guanidine lysis buffer containing 6M guanidinehydrochloride 20mM sodium phosphate 500mM NaCland pH 78 The cell suspension was sonificated (three burstsof 1min each at 1min interval) After 30ndash60min incubationin ice with slight shaking the cell lysate was centrifugedat 13000 rpm and 4∘C for 25min to remove cell debris

BioMed Research International 3

A volume of 8mL cell lysate was applied to a Ni-NTA column(Invitrogen Corp Carlsbad USA) containing 2mL resinwhich was equilibrated with denaturing binding buffer andincubated for 45min at room temperature with gentle handshaking for several times The column was washed with 4times of 8mL denaturing wash bufferThe bound protein waseluated with 8mL of denaturing eluation buffer Then 6mLof the enzyme extract was applied to a Bio-gel column (26 times6 cm) with elution of 50mM Tris-HCl buffer (pH 8) at a flowrate of 25mLh and then washed with the same buffer

27 Streptokinase Renaturation The pool of purified SKfragments were renaturated using 50mM phosphate bufferpH 7 supplemented with 10 (wv) glycerol and differentdetergents (05 (wv) Triton X-100 1 (wv) Tween 20 15(wv) Tween 80) [11] Diluted cell lysate (1 200) and purifiedrSK (1 100) in renaturation buffer were incubated at 37∘C for1 h and 4∘C for 6 hThe residual activity was then determinedas described below

28 Streptokinase Assay To estimate the activity of thepurified rSK 10 120583L purified protein solution was added to10 120583L of 50mM Tris buffer pH 75 containing 005 unit ofhuman plasminogen and incubated at 37∘C for 30min Thecolor reactionwas developed by the addition of 40120583Lof 1mMAAS solution and incubated at 37∘C for 15min The reactionwas stopped by the addition of 10 120583L of 04 N acetic acid Theabsorbance was read at 405 nm against a blank containinghuman plasminogen Tris buffer and AAS but without rSKsolutionThe activitywas estimated using standard SK (SigmaAldrich Co St Luis USA) One unit (U) of rSK was definedas one unit of standard SK which liquefies a standard clot offibrinogen plasminogen and thrombin at 37∘Cand pH75 for10min

29 Protein Electrophoresis and Quantification The homo-geneity and molecular mass of the streptokinase were deter-mined by 125 SDS polyacrylamide gel electrophoresis [16]with Biometra equipment (Gottingen Germany) Proteinswere visualized by staining with Coomassie Brilliant Blue R-250 orwith 01 (wv) of silver nitrate Protein concentrationswere measured by Bradford assay with the bovine serumalbumin as standard [17]

210MALDI-TOFMass Spectrometry The rSKwas identifiedby MALDI-TOF mass spectrometry as previously described[18] The predicted protein band on SDS-PAGE was cut outand the target protein was digested by trypsin treatment intosmall peptide fragments The mixture of peptides was ana-lyzed on nano-LC liquid chromatography and ionized by theESI (electrospray ionization)Themass spectra were obtainedby QSTAR XL mass spectrometer (Applied BiosystemsMDS SCIEX Canada) with a nano-ESI ion source Proteinfragments were identified by theMascot v18 Search Softwarefrom the database (NCBInr SwissProt) Peptide fragmentsshowing ion scores above 42were identified uniquely or high-similarly with 119875 lt 005

211 Biochemical Characterization of rSK The pH and tem-perature optimum of rSK were determined by measuringthe activity as described above using 100mM potassiumphosphate buffer (pH 55ndash75) and 100mM Tris-HCl buffer(pH 75ndash10) at 37∘C and in the temperature range of 4to 60∘C using 100mM potassium phosphate buffer pH 75respectively

For the determination of temperature and pH stabilitythe purified rSK 01120583g for each reaction was preincubatedin 100mM potassium phosphate buffer pH 7 at differenttemperatures 4ndash60∘C for 0ndash96 h and pH range from 4 to95 (pH 4-5 100mM potassium acetate buffer pH 55ndash75100mMpotassiumphosphate buffer and pH 75ndash95 100mMTris-HCl) at 37∘C for 0ndash48 h respectively The residualactivity was then determined

Effect of surfactants on the activity of rSK was checkby mixture of 04 unit purified rSK and substrate andsupplemented with either Triton X-100 Tween 20 or Tween80 each at a final concentration of 05 10 15 and 20 (wv)in appropriate buffer pH 7 and incubated at 37∘C for 60minThe residual activity of rSK was determined as describedabove

The effect of additives on the activity of the purified rSKwas investigated by incubating the mixture of 04 unit of thepurified rSK and either of Ag+ Ca2+ Co2+ Cu2+ Fe2+ K+Mn2+ Ni2+ Zn2+ or EDTA at a final concentration of 1 3and 5mMThe reaction mixtures were incubated at 28∘C for60min The residual activity of rSK was then measured asshown above All measurements were carried out in triplicatewith the resulting values being the mean of the cumulativedata obtained

3 Results and Discussion

31 Gene Cloning and Analysis The recombinant plasmidpTSK with inserted sk gene was sequenced and alignedwith sequences from GenBank using DNAstar Nucleotidesequence of sk gene from S pyogenesDT7 exhibited 844 to996 identities with sequences from Streptococcus pyogenesgroups of A C and G strains in GenBank (CP000262CP000261 M19347 AM903378 and AY234136) The putativeamino acid sequence of the gene sk showed 779 to 993identities with the corresponding amino acid sequencesfrom the abovementioned Streptococcus pyogenes strainsThesequence was deposited in the GenBank with an accessionnumber of ACG50170

32 Expression and Purification of SK The DNA fragment(1245 bps) encoding the mature streptokinase (SK) truncated26 N-terminal amino acids from S pyogenes DT7 wasinserted into pET22b+ vector at the BamHI and XhoI sitesresulting in the recombinant plasmid pESK The transfor-mant E coli BL21pESK was grown in LB medium for the SKproduction After IPTG induction the cells were collectedand used for purification and renaturation The expressionlevel of rSK as inclusion bodies by E coli BL21pESK was60 of the total proteins (Figure 1(a) lane 1) using Dolphin1D software This level was as high as that (65) reported by


(kDa) 2M1

66

45

35

25

18

116

14

(a)

3M(kDa)

66

45

35

25

18

116

(b)

Figure 1 SDS-PAGE of the purified rSK by ProBond Resin Lane 1 E coli BL21pESK cell lysate Lane 2 purified rSK stained by usingCoomassie Brilliant Blue R250 Lane 3 purified rSK stained by using silver nitrate Lane M molecular standards indicated in kDa

Table 1 Effect of surfactant glycerol and temperature on the renaturation of cell lysate E colipESK

Parameter SK activity (UmL)at 37∘C for 1 h at 37∘C for 1 h + 10 glycerol at 4∘C for 6 h + 10 glycerol

05 Triton X-100 6434 plusmn 36 55740 plusmn 362 75913 plusmn 4521 Tween 20 6576 plusmn 57 45928 plusmn 271 65877 plusmn 27115 Tween 80 6916 plusmn 66 52268 plusmn 506 65787 plusmn 633No surfactant 1821 plusmn 18 1843 plusmn 36 1894 plusmn 36

Table 2 The renaturation of purified rRSK

Fraction number rSK activity (Umg)at 37∘C for 1 h at 4∘C for 6 h + 05 Triton X-100 and 10 glycerol at 37∘C for 1 h + 05 Triton X-100 and 10 glycerol

1 691 plusmn 10 16224 plusmn 293 25190 plusmn 1572 3605 plusmn 48 103125 plusmn 552 112642 plusmn 2763 1999 plusmn 14 20393 plusmn 211 38625 plusmn 1524 553 plusmn minus03 15169 plusmn 189 19076 plusmn 118

Zhang et al (1999) [19] and more than two to four times ashigh as those (25) reported by [20] (20) by [21] and 15by [22]

33 Renaturation of Streptokinase The cell lysate was rena-tured by using various surfactants including Triton X-100Tween 20 and Tween 80 each or in combination with glyc-erol Triton X-100 was known as detergent to dissolves andrefolding aggregated protein In absence of surfactants rSKexhibited the same activity (182ndash189UmL) with or withoutglycerol (Table 1) The addition of surfactants increased therSK activity obviously to 35ndash38 folds without glycerol butsteeply to 252ndash306-folds in combination with glycerol at37∘C for 60min even to 361ndash417 folds at 4∘C for 6 h At

lower temperature (4∘C) the enzyme activity was recoveredbetter than at higher temperature (37∘C) increased by 26ndash43The combination of glycerol at the concentration of 10(wv) and Triton X-100 at the concentration of 05 (wv)recovered the highest activity of rSK and reached 7591UmLat 4∘C (Table 1) The renaturation of the purified rSK with10 glycerol containing 05 Triton X-100 at 4∘C for 6 hand at 37∘C for 1 h recovered the enzyme activity of 286and 365 folds respectively (Table 2) corresponding to thespecific activity of 103125 and 112642Umg protein Thereason the renaturation efficiency in this study was muchhigher than that reported by Cherish Babu et al (2008) Atthe same conditions for treatment the enzyme activity wasrecovered with only 97 folds in comparison to control [11]


Table 3 Purification steps of the streptokinase from E colipESK

Total proteins Purified proteins Specific activity (Umg) of Yield () Purification factorsupernatant of cell lysate purified rSK

90375mg 21789mg 4568 10336 52 226

34 Purification of Recombinant SK rSK from S pyogenesDT7 overexpressed by E coli BL21pESK cells was purifiedthrough affinity chromatography column of Ni2+-ProBondresin to the homogeneity on SDS-PAGE with a molecularmass of approximately 47 kDa (Figure 1 lane 2) The purifiedrSak gained a specific activity of 10336Umg proteins witha purification factor of 256 and a yield of 52 (Table 3)The solution containing rSK protein was loaded onto BiogelP-100 packed column for fractionating and obtained with apurity of 957 and specific activity of 11558Umg (Figure 1lane 3)

35 Identification of Recombinant SK The single proteinon SDS-PAGE (Figure 1 lane 3) was cut out from the geland used for LC-ESI-MSMS analysis of mass spectrumdatabase by using Mascot v18 program The total scoreof SK identification was 203 to 509 and matched peptideswere 29 to 39 fragments Four peptide fragments ofthe purified enzyme identified by MALDI-TOF massspectrometry agreed with those of the streptokinasefound in GenBank gi|153807 streptokinase (S pyogenes)VNVNYEVSFVSETGDLDFTPLLR (position 158ndash180)(Figure 2(a)) NQYHLTTLAVGDSLSSQELAAIAQFILSK(position 181ndash209) (Figure 2(b)) TNNTDLISEKYYVLK(position 263ndash278) (Figure 2(c)) NLDFRDLYDPR (position320ndash330) (Figure 2(d)) corresponding to a monoisotopicmass of 26133 311763 179993 and 142269Da and to mzion scores of 102 111 73 and 51 respectively Whereasthe peptide fragments showing ion scores above 42 wereidentified uniquely or highly similarly to 119875 lt 005 Thesepeptides of the recombinant streptokinase expressed by EcolipESK showed 100 identity with the correspondingfragments of the putative streptokinase protein from Spyogenes (gi|153807) (Figure 2(e))

36 Temperature and pHOptimum The temperature and pHoptimum for the reaction of SK-plasmin were observed at37∘C and pH 7 (Figures 3(a) and 3(b)) The enzyme showedover 80 activity at the temperature range from 25 to 45∘Cand pH 67ndash75 (for 100mM potassium phosphate buffer)and pH 85ndash10 (for Tris-HCl buffer) in comparison withthe optimum activity The temperature optimum for the SK-plasmin reaction was in agreement with that from otherreports Rajagopalan et al (1987) reported that the reactionsof 1205722-macroglobulin (1205722M) with plasmin or streptokinase-plasmin (ogen) (SkP1) was markedly temperature-dependentand initial rates of reaction at 0 and 24∘C were only 3 and40 of the rate of 37∘C respectively [23] Mumme et al(1993) reported that the highest fibrinolysis activity withstreptokinase was obtained at 40∘C with lower activities

having been recorded at both higher and lower temperatures[24]Theoptimum temperature andpHof streptokinase from120573-haemolytic streptococci were 27ndash37∘C and 7 [25] Anotherthrombolytic agent closely related to the streptokinasestaphylokinase (Sak) from Staphylococcus aureus exhibitedthe same profile The native Sak from S aureus V8 showedthe pH optimum at pH 75 and 85 [26] The temperature andpH optimum for Sak from S aureusQT08 expressed in E coliand P pastoriswere observed at 30ndash37∘C pH 7 and pH 9 [27]and pH 75 and pH 85 [28] respectively

Why the streptokinases and staphylokinases shared acommon property that the optimum temperature was notmore than 40∘C and pH optimum exhibited 2 peaks becausethe fibrinolytic activity of streptokinase originates in its abilityto activate blood plasminogen to plasmin the enzyme thatdegrades fibrin cloth through its specific lysine binding site[29] The temperature optimum for the human plasmin wasat 37∘C [30] and the optimal pH value for the human plasminand that for SK or Sak were significantly different

37 Temperature and pH Stability The streptokinase from Spyogenes DT7 was stable up to 37∘C and retained more than80 of its initial activity after incubation for 9 h and morethan 50 after incubation for 96 h (Figure 4(a)) The enzymeexhibited more stability at pH 7 than at pH 9 and retainedmore than 73 of its initial activity after incubation at pH7 for 24 h whereas it retained only more than 65 of itsinitial activity after incubation at pH 9 for 8 h (Figure 4(b))K Vesterberg and O Vesterberg (1972) also reported thatthe concentrated material containing Sak from S aureus V8was stable at refrigerator temperature over a pH range of30ndash85 Sak from S aureus QT08 expressed in E coli andP pastoris was stable at a temperature range from 25∘C to50∘C and at a pH range from 7 to 9 after incubation for 2 hwith a residual activity of more than 70 [26 28]The resultsdepicted in Figure 4(b) indicating that there were two sharppeak one at pH 70 and the other one at pH 90 with theactivity of 100 and 98 respectivelyThe experiments of theoptimal pH value for the high level activity of rSKwere rathercomplicated since two reactions happened continuously inthe same reaction mixture at first the activation reaction ofplasminogen to plasmin was activated by rSK and secondthe digestion process of AAS was catalyzed by plasminThe optimal pH value for human plasmin and that for SKwas significantly different therefore this could cause theappearance of second peak activity The data depicted inFigure 4(b) showing that the second peak activity at pH valueof 90 might therefore be due to optimal pH for the plasminactivity in Tris-HCl buffer Similarly these observations werealso reported by K Vesterberg and O Vesterberg (1972) inwhich staphylokinase was a plasminogen activator


200 400 600 800 1000 1200 1400 1600 1800

y(1)

b(4)++

b(3)

ylowast(4)y(4)

y(5)

blowast(6)

y(6)

y(7)

y(8)

y(9) y(10)

y(11)

y(12)

y(14)

y(17)

(a) VNVNYEVSFVSETGDLDFTPLLR

200 400 600 800 1000 1200 1400 1600

b(5)++

blowast(3)

a(7)++

y(8)++

b(8)++

blowast(9)++ b(9)++

b(10)++

y(5)

a(5)

b(4)

b(5)

y(13)++

y(6)

blowast(6)

b(14)++

y(7)

blowast(7)

b(7)

y(8)

blowast(8)

b(18)++

alowast(9)alowast(19)++

blowast(9)

y(10)

y(9)

blowast(10)

b(10)

y(11)

b(11)

ylowast(12)blowast(12)y(12)

b(12)

blowast(13)b(13)

ylowast(13)y(13)

b(28)++blowast(14)b(14)

y(14)

blowast(15)b(15)

y(15)

blowast(16)

y(3)

y(2)

alowast(4)

a(19)++

(b) NQYHLTTLAVGDSLSSQELAAIAQFILSK

200 400 600 800 1000 1200 1400 1600 1800

ylowast(1) y(1)

blowast(2) b(4)++

y(2)

blowast(3)b(3)

b(12)

y(3)

alowast(5) y(8)++y(4)

y(9)++

blowast(6)

y(11)++

y(12)++

ylowast(13)++

y(13)++ a(14)++ y(14)++

y(7)

ylowast(8) y(8)

y(9)

y(10)

y(11)

(c) TNNTDLISEKYYVLK

200 400 600 800 1000 1200 1400 1600

b(1)

ylowast(1) y(1)a(2)

b(2)

ylowast(2) y(2)

b(3)

ylowast(3)

b(4)

ylowast(8)++

y(8)++

blowast(5) b(5)

y(5)

blowast(6) b(6)

a(7)blowast(7)b(7)ylowast(7)

alowast(8) a(8)

b(8)ylowast(8) y(8)

blowast(9) b(9)

ylowast(9)

ylowast(10)

(d) NLDFRDLYDPR

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+10 1009080706050403020

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+1 GI153807aIAGYEWLLDRPSVNNSQLVVSVAGTVEGTNQEISLKFFEIDLTSRPAHGGKTEQGLSPKSKPFATDKGAMSHKLEKADLLKAIQEQLIANVHSNDGYFEV1 ---------------------------------------------------------------------------------------------------- 4 peptides1 ACG50170aIAGPEWLLDRPSVNNSQLVVSVAGTVEGTNQEISLKFFEIDLTSGPAQGGKTEQGLSPKSKPFATNKGAMPHKLEKADLLKAIQEQLIANVHSNDGYFEV

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+110 200190180170160150140130120

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+101 GI153807aIDFASDATITDRNGKVYFADKDDSVTLPTQPVQEFLLSGHVRVKPYQPKAVHNSAERVNVNYEVSFVSETGDLDFTPLLRNQYHLTTLAVGDSLSSQELA

1 ---------------------------------------------------------VNVNYEVSFVSETGDLDFTPLLRNQYHLTTLAVGDSLSSQELA 4 peptides101 IDFASDATITDRNGKVYFADRDDSVTLPTQPVQEFLLSGHVRVRPYQPKAVHNSAERVNVNYEVSFVSETGDLDFTPLLRNQYHLTTLAVGDSLSSQELA ACG50170a

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+210 240230220 250 300290280270260

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+201 AIAQFILSKKHPDYIITKRDSSIVTHDNDIFRTILPMDQEFTYHIKDREQAYKANSKTGIEEKTNNTDLISEKYYVLKKGEKPYDPFDRSHLKLFTINYV GI153807a

44 AIAQFILS------------------------------------------------------KTNNTDLISEKYYVLK---------------------- 4 peptides201 ACG50170aAIAQFILSKKYPDYIITKRDSSIVTHDNDIFRTILPMDQEFTYHIKDREQAYKANSKTGIVEKTNNTDLISEKYYVLKKGEKPYDPFDRSHLKLFTINYV

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+310 400390380370360350340330320

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+301 GI153807aDVNTNKLLKSEQLLTASERNLDFRDLYDPRDKAKLLYNNLDAFGIMDYTLTGKVEDNHDDTNRIITVYMGKRPEGENASYHLAYDKDRYTEEEREVYSYL

68 -------------------NLDFRDLYDPR 4 peptides301 ACG50170aDVNTNELLKSEQLLTASERNLDFRDLYDPRDKAKLLYNNLDAFGIMDYTLTGKVEDNHNDTNRIITVYMGKRPEGENASYHLAYDKDRYTEEEREVYSYL

---------+----410

---------+----401 GI153807aRYTGTPIPDNPKDK

78 4 peptides401 RYTGTPIPDNPKDK ACG50170a

(e)

Figure 2 Monoisotopic mass of three neutral identified peptides (a) VNVNYEVSFVSETGDLDFTPLLR position 158ndash180 (a) (b)NQYHLTTLAVGDSLSSQELAAIAQFILSK position 181ndash208 (c) TNNTDLISEKYYVLK position 263ndash279 (d) NLDFRDLYDPR position320ndash330 found in gi 153807 streptokinase from Streptococcus pyogenes (GenBank AAA26973) corresponding to ion scores of 102 111 73and 51 with 119875 lt 005 respectively (e) Alignment of four neutral identified peptides (4 peptides) with streptokinase from Streptococcuspyogenes AAA26973 (gi153807) and rSK from S pyogenes DT07 (ACG50170)


0

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0 10 20 30 40 50 60 70

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tive S

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Figure 3 Temperature (a) and pH (b) optimum of rSK from S pyogenes DT07

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()

Incubation time (h)

4∘C25

∘C37

∘C

45∘C

60∘C

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120 Re

lativ

e SK

activ

ity (

)

Incubation time (h) pH 7

pH 9

(b)

Figure 4 Temperature (a) and pH (b) stability of rSK from S pyogenes DT07

38 Effect of Surfactants The addition of either Tween 80Tween 20 or Triton X-100 at the final concentration of05ndash2 (wv) in reaction mixture showed an activation ofthe streptokinase from S pyogenes DT07 up to 150 of itsoriginal activity The enzyme activity increased up to 154after incubation for 24 h but deeply decreased to 18 afterlonger incubation for 48 h (Table 4) Similarly Cherish Babuet al (2008) reported that rSK was treated with guanidineand then supplemented with Triton X-100 that enhanced theactivity of rSK

39 Effect of Metal Ions and EDTA In the present studyeffect of various additives on the purified rSK activity wasinvestigated The addition of EDTA and metal ions showed aclear effect on the streptokinase activity EDTAMn2+ andK+inhibited the enzyme partially whereas Ag+ Ca2+ and Co2+exhibited a strong inhibition But the metal ions Cu2+ Fe2+Ni2+ and Zn2+ at a final concentration of 1mM completely

inhibited the streptokinase (Table 5) In previous studiesit was also observed that the addition of Zn2+ and Cu2+almost completely inhibited the activity of the recombinantstaphylokinase from Staphylococcus aureus QT08 [27] andthe native staphylokinase from S aureus V8 [31] anotherthrombolytic agent closely related to the streptokinase Whythe streptokinases and staphylokinases shared a commonproperty that addition of Zn2+ and Cu2+ resulted in almostcompletely inhibition of activities Because the plasmincompletely lost its activity when it was incubated with Zn2+and Cu2+ [32 33]

4 Conclusion

SK is a promising blood-clot dissolving agent for the treat-ment of patients suffering from a heart attack It would bedesirable to produce high yield of protein with high activityfor thrombolytic therapy In the present study a sk gene from


Table 4 Effect of surfactants on streptokinase activity

Detergent Residual activity () at the concentration ()05 10 15 20

Tween 20 1375 plusmn 26 1383 plusmn 18 1350 plusmn 23 1365 plusmn 26Tween 80 1191 plusmn 28 1259 plusmn 26 1422 plusmn 15 1154 plusmn 22Triton X-100 1503 plusmn 19 1406 plusmn 36 1436 plusmn 09 1266 plusmn 20

Relative activity () after incubation for (h)0 8 24 48


Table 5 Effect of metal ions on streptokinase activity

Additive Residual activity () at the concentration (mM)1 3 5

AgNO2 161 plusmn 01 nd ndCaCl2 145 plusmn 02 nd ndCoCl2 156 plusmn 01 nd ndEDTA 504 plusmn 02 705 plusmn 03 882 plusmn 04KCl 185 plusmn 01 252 plusmn 02 596 plusmn 05MnSO4 405 plusmn 04 542 plusmn 03 684 plusmn 05

Streptococcus pyogenesDT7 was overexpressed in E coli witha level of 60 of total proteins which is highest yield of anyrSK expressed in E coli till date A simple renaturation systemdramatically covered the rSK activity with 41 folds whichwas not reported before Overproduction of rSK in E coli incombination with a simple and highly effective renaturationmade the recombinant E coli become a potential strain forindustrial SK production

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the Ministry of Science andTechnology with the project KC102806-10 ldquoProduction ofRecombinant Streptokinase and Tissue Plasminogen Activa-tor Used for Therapyrdquo and Vietnam Academy of Science andTechnology (2008)

References

[1] R N Vellanki R Potumarthi and L N Mangamoori ldquoCon-stitutive expression and optimization of nutrients for streptok-inase production by Pichia pastoris using statistical methodsrdquoApplied Biochemistry and Biotechnology vol 158 no 1 pp 25ndash40 2009

[2] V Kunadian and CM Gibson ldquoThrombolytics andmyocardialinfarctionrdquo Cardiovascular Therapeutics vol 30 no 2 pp e81ndashe88 2012

[3] C Klessen and H Malke ldquoExpression of the streptokinase genefrom Streptococcus equisimilis in Bacillus subtilisrdquo Journal ofBasic Microbiology vol 26 no 2 pp 75ndash81 1986

[4] H Malke D Gerlach W Kohler and J J Ferretti ldquoExpressionof a streptokinase gene from Streptococcus equisimilis in Strep-tococcus sanguisrdquo Molecular and General Genetics vol 196 no2 pp 360ndash363 1984

[5] E Pimienta J C Ayala C Rodrıguez et al ldquoRecombinantproduction of Streptococcus equisimilis streptokinase by Strepto-myces lividansrdquoMicrobial Cell Factories vol 6 article 20 2007

[6] M-R Kim Y-H Choeng W-J Chi D-K Kang and S-KHong ldquoHeterologous production of streptokinase in secretoryform in Streptomyces lividans and in nonsecretory form inEscherichia colirdquo Journal of Microbiology and Biotechnology vol20 no 1 pp 132ndash137 2010

[7] R Kumar and J Singh ldquoExpression and secretion of a prokary-otic protein streptokinase without glycosylation and degrada-tion in Schizosaccharomyces pomberdquo Yeast vol 21 no 16 pp1343ndash1358 2004

[8] J Pratap and K L Dikshit ldquoEffect of signal peptide changeson the extracellular processing of streptokinase from Echerichiacoli requirement for secondary structure at the cleavage junc-tionrdquo Molecular and General Genetics vol 258 no 4 pp 326ndash333 1998

[9] K Sriraman and G Jayaraman ldquoEnhancement of recombinantstreptokinase production in Lactococcus lactis by suppression ofacid tolerance responserdquo Applied Microbiology and Biotechnol-ogy vol 72 no 6 pp 1202ndash1209 2006

[10] H Malke and J J Ferretti ldquoStreptokinase cloning expressionand excretion by Escherichia colirdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 81 no11 pp 3557ndash3561 1984

[11] P V Cherish Babu V K Srinivas V Krishna Mohan andE Krishna ldquoRenaturation purification and characterization of


streptokinase expressed as inclusion body in recombinant Ecolirdquo Journal of ChromatographyBAnalytical Technologies in theBiomedical and Life Sciences vol 861 no 2 pp 218ndash226 2008

[12] J Pratap G Rajamohan and K L Dikshit ldquoCharacteristicsof glycosylated streptokinase secreted from Pichia pastorisenhanced resistance of SK to proteolysis by glycosylationrdquoApplied Microbiology and Biotechnology vol 53 no 4 pp 469ndash475 2000

[13] F Baneyx ldquoRecombinant protein expression inEscherichia colirdquoCurrent Opinion in Biotechnology vol 10 no 5 pp 411ndash4211999

[14] B Balagurunathan and G Jayaraman ldquoTheoretical and exper-imental investigation of chaperone effects on soluble recombi-nant proteins in Escherichia coli effect of free DnaK level ontemperature-induced recombinant streptokinase productionrdquoSystems and Synthetic Biology vol 2 no 1-2 pp 27ndash48 2008

[15] D T Quyen T T Dao and S L T Nguyen ldquoA novelesterase from Ralstonia sp M1 gene cloning sequencing high-level expression and characterizationrdquo Protein Expression andPurification vol 51 no 2 pp 133ndash140 2007

[16] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[17] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[18] T T H Vu D T Quyen T T Dao and S L T NguyenldquoCloning high-level expression purification and propertiesof a novel endo-120573-14-mannanase from Bacillus subtilis G1 inPichia pastorisrdquo Journal of Microbiology and Biotechnology vol22 no 3 pp 331ndash338 2012

[19] X-W Zhang T Sun X-N Huang X Liu D-X Gu andZ-Q Tang ldquoRecombinant streptokinase production by fed-batch cultivation of Escherichia colirdquo Enzyme and MicrobialTechnology vol 24 no 10 pp 647ndash650 1999

[20] N Perez E Urrutia J Camino et al ldquoHydrophobic interactionchromatography applied to purification of recombinant strep-tokinaserdquo Minerva Biotecnologica vol 10 no 4 pp 174ndash1771998

[21] B Balagurunathan N S Ramchandra and G JayaramanldquoEnhancement of stability of recombinant streptokinaseby intracellular expression and single step purification byhydrophobic interaction chromatographyrdquo BiochemicalEngineering Journal vol 39 no 1 pp 84ndash90 2008

[22] E Pupo B A Baghbaderani V Lugo J Fernandez R Paezand I Torrens ldquoTwo streptokinase genes are expressed withdifferent solubility in Escherichia coli W3110rdquo BiotechnologyLetters vol 21 no 12 pp 1119ndash1123 1999

[23] S Rajagopalan S L Gonias and S V Pizzo ldquoThe temperature-dependent reaction between alpha 2-macroglobulin andstreptokinase-plasmin(ogen) complexrdquo The Journal ofBiological Chemistry vol 262 no 8 pp 3660ndash3664 1987

[24] A Mumme M Kemen H-H Homann and V Zumto-bel ldquoTemperature-dependent fibrinolysis with streptokinaserdquoDeutsche Medizinische Wochenschrift vol 118 no 44 pp 1594ndash1596 1993

[25] R Dubey J Kumar D Agrawala T Char and P PuspldquoIsolation production purification assay and characterizationof fibrinolytic enzymes (Nattokinase Streptokinase and Uroki-nase) from bacterial sourcesrdquo African Journal of Biotechnologyvol 10 no 8 pp 1408ndash1420 2011

[26] K Vesterberg and O Vesterberg ldquoStudies of staphylokinaserdquoJournal of Medical Microbiology vol 5 no 4 pp 441ndash450 1972

[27] T H T Nguyen and D T Quyen ldquoCloning high-level expres-sion purification and characterization of a staphylokinasevariant Sak120593C from Staphylococcus aureusQT08 in Escherichiacoli BL21rdquo African Journal of Biotechnology vol 6 pp 2129ndash2136 2012

[28] TH TNguyen andD TQuyen ldquoHigh-level expression purifi-cation and properties of a fully active even glycosylated staphy-lokinase variant Sak120593C from Staphylococcus aureus QT08 inPichia pastorisrdquo African Journal of Microbiology Research vol11 pp 5995ndash6003 2012

[29] P Rodriguez P Fuentes M Barro et al ldquoStructural domainsof streptokinase involved in the interaction with plasminogenrdquoEuropean Journal of Biochemistry vol 229 no 1 pp 83ndash90 1995

[30] H Lu C Soria E M Cramer et al ldquoTemperature dependenceof plasmin-induced activation or inhibition of human plateletsrdquoBlood vol 77 no 5 pp 996ndash1005 1991


[32] L I Sokolovskaya A Y Slominskii and G L Volkov ldquoInduc-tion of catalytic activity of plasminogen by monoclonal anti-body IV-Ic in the presence of divalent metal cations and 1205722-antiplasminrdquo Biochemistry vol 71 no 6 pp 627ndash633 2006

[33] P Nowak and A Zgirski ldquoEffects of metal ions on activity ofplasminrdquo Biological Trace Element Research vol 93 no 1-3 pp87ndash94 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


characteristics of recombinant products and the effect of theexpressed foreign protein on host cell physiology [14]

The expression of SK as inclusion bodies byE coli systemsis shown to be useful for obtaining large amounts of proteinprovided that renaturation is effective and recovery of activeprotein is high Thus the purpose of this study was firstlyto overproduce the recombinant streptokinase in E coliBL21 (DE3) and simultaneously to refold effectively the largeamount of the recombinant streptokinase as inclusion bodiesoverexpressed by E coli BL21 (DE3) under the control ofthe promoter T7 Only both objectives were gained then therecombinant E coli overproducing SK as inclusion bodiescan become a potential strain for industrial SK produc-tion

2 Materials and Methods

21 Chemicals and Reagents DNA cloning kit RNase Arestriction enzymes (BamHI NotI and EcoRI) T4-ligaseand Proteinase K were purchased from Fermentas (ThermoFisher Scientific Inc Waltham USA) The DNA ExtractionKit was from Qiagen (Venlo Netherlands) Protein Extrac-tion Kit and ProBond resin were supplied by InvitrogenCorp (Carlsbad CA USA) Human plasminogen from MPBiomedicals (Santa Ana USA) SK N (p-tosyl) gly-pro-lys-4-nitro anilide acetate salt (AAS) SDS from Sigma Aldrich Co(St Luis USA) Plasminogen Tween 20 and Tween 80 fromBioBasic Inc (NYUSA) TritonX-100 andEDTA fromMerck(Darmstadt Germany) All other reagents were of analyticalgrade unless otherwise stated

22 Plasmids Bacterial Strains and Culture Conditions Thebacterial strain Streptococcus pyogenes DT7 (GQ247718) iso-lated from a patient at the Army Hospital No 103 (HanoiVietnam) was used as the source of the streptokinase (sk)gene Escherichia coli DH5120572 (Fminus oslash80dlacZΔM15 Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17(rKminus mK+) phoAsupE44 120582ndash thi-1 gyrA96 relA1) and the vector pJET12blunt(Fermentas Thermo Fisher Scientific Inc Waltham USA)were used for DNA manipulations and amplificationEscherichia coli BL21 (DE3) cells (FndashompT gal dcm lon hsdSB(119903119861

minus119898119861

minus) 120582(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])and pET22b+ vector (Novagen Merck KGaA DarmstadtGermany)were used for expression of SK LBmedium (Luria-Bertani) containing 1 (wv) bacto tryptone 05 (wv) yeastextract 1 (wv) NaCl pH 7ndash75 was used for cultivation ofE coliDH5120572 and BL21 (DE3) LB agar contained additionally2 (wv) agar and 100 120583g ampicillinmL

23 DNA Manipulations Genomic and plasmid DNA isola-tion was carried out by methods which have been previouslydescribed [15] DNA fragments and PCR products wereexcised from a 08 agarose gel and purified by a gelextraction kit (Qiagen Venlo The Netherlands) accordingto the manufacturerrsquos instructions DNA sequencing wasperformed on an ABI PRISM 3100 Avant Genetic Analyzer(Applied Biosystems Inc Foster City USA) E coli DH5120572

and BL21 were transformed using heat shockmethod that hasbeen previously described [15]

24 DNA Amplification and Plasmid Construction The puta-tive sk-coding DNA fragment was amplified from S pyogenesDT7 genomic DNA by PCR with Taq DNA polymeraseBased on the nucleotide sequence of the sk gene fromS pyogenes strain (GenBank Z48617) 3 oligonucleotidesmSKF GGC GGATCC CATATG ATTGCTGGACCTG andSKF GCC CAT GGG CAA AAA TTA CTT AT and SKRGCC TCG AGT TTG TCB TTA GGG TT were designed asprimers for introduction of the underlined BamHI and XhoIrestriction sites respectively The PCR mixture contained25 120583L 10x PCR buffer 2 120583L of 25mMdNTP 25 120583L of 25mMMgCl

2

05 120583L genomic DNA (50ndash100 ng) 025120583L 5 unit Taqpolymerase and 1 120583L each primer (10 pmol) supplementedwith 1525120583L distillated water to a final volume of 25120583L Thethermocycler conditions were as follows 95∘C41015840 30 cycles of(95∘C3010158401015840 52∘C4510158401015840 72∘C4510158401015840) 72∘C101015840The PCR productsamplified from the genomic DNA with the primer pair SKFand SKR were inserted into the cloning vector pJET12bluntresulting in pJSK DNA sequencing was performed on ABIPRISM 3100 Avant Genetic Analyzer Sequence alignmentswere constructed and analyzed using the programMegAlignDNAStar It was followed by ligation of the BamHI-XhoIdigested PCR products (with the primer pairmSKF and SKR)with pET22b+ linearized by the same enzymes resultingin pESK under the control of the T7-promoter induced byisopropyl-120573-D-thiogalactopyranoside (IPTG) and possessingthe ampicillin marker The streptokinase rSKhis encodedby the plasmid pESK contains the mature streptokinasefused with the 6x histidine-tag and no native leader seq-uence

25 rSK Expression The transformant E coli BL21pESK wascultivated overnight in 5mL of LB medium containing 5120583Lof 100mgmL ampicillin at 37∘C on an orbital shaker at200 rpm Overnight culture (2mL) was inoculated in a 1-liter Erlenmeyer flask containing 200mL of LB broth and200120583L of 100mgmL ampicillin The culture was grownat 37∘C with agitation at 200 rpm and until an opticaldensity (OD) at 600 nm reached 06 (for approximately 25 h)then 200 120583L of 100mM IPTG was added The culture wascontinuously incubated at 37∘C with agitation at 200 rpm for3ndash6 h induction Cells were harvested by centrifugation at6000 rpm for 10min at 4∘C Wet weight cells were used forprotein purification

26 Purification of Streptokinase The fusion form rSKhis car-rying a C-terminal 6xHis tag was expressed in E coli BL21 Topurify rSK 100mg wet weight cells from a 120mL culture inLB medium were harvested by centrifugation and suspendedin 10mL of guanidine lysis buffer containing 6M guanidinehydrochloride 20mM sodium phosphate 500mM NaCland pH 78 The cell suspension was sonificated (three burstsof 1min each at 1min interval) After 30ndash60min incubationin ice with slight shaking the cell lysate was centrifugedat 13000 rpm and 4∘C for 25min to remove cell debris















(kDa) 2M1

66

45

35

25

18

116

14

(a)

3M(kDa)

66

45

35

25

18

116

(b)














90375mg 21789mg 4568 10336 52 226








200 400 600 800 1000 1200 1400 1600 1800

y(1)

b(4)++

b(3)

ylowast(4)y(4)

y(5)

blowast(6)

y(6)

y(7)

y(8)

y(9) y(10)

y(11)

y(12)

y(14)

y(17)


200 400 600 800 1000 1200 1400 1600

b(5)++

blowast(3)

a(7)++

y(8)++

b(8)++

blowast(9)++ b(9)++

b(10)++

y(5)

a(5)

b(4)

b(5)

y(13)++

y(6)

blowast(6)

b(14)++

y(7)

blowast(7)

b(7)

y(8)

blowast(8)

b(18)++


blowast(9)

y(10)

y(9)

blowast(10)

b(10)

y(11)

b(11)


b(12)

blowast(13)b(13)

ylowast(13)y(13)

b(28)++blowast(14)b(14)

y(14)

blowast(15)b(15)

y(15)

blowast(16)

y(3)

y(2)

alowast(4)

a(19)++


200 400 600 800 1000 1200 1400 1600 1800

ylowast(1) y(1)

blowast(2) b(4)++

y(2)

blowast(3)b(3)

b(12)

y(3)


y(9)++

blowast(6)

y(11)++

y(12)++

ylowast(13)++

y(13)++ a(14)++ y(14)++

y(7)

ylowast(8) y(8)

y(9)

y(10)

y(11)

(c) TNNTDLISEKYYVLK

200 400 600 800 1000 1200 1400 1600

b(1)

ylowast(1) y(1)a(2)

b(2)

ylowast(2) y(2)

b(3)

ylowast(3)

b(4)

ylowast(8)++

y(8)++

blowast(5) b(5)

y(5)

blowast(6) b(6)


alowast(8) a(8)

b(8)ylowast(8) y(8)

blowast(9) b(9)

ylowast(9)

ylowast(10)

(d) NLDFRDLYDPR

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+10 1009080706050403020


---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+110 200190180170160150140130120



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+210 240230220 250 300290280270260



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+310 400390380370360350340330320



---------+----410



(e)



0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Rela

tive S

K ac

tivity

()

Temperature (∘C)

(a)

Rela

tive S

K ac

tivity

()

120

100

80

60

40

20

0

5 6 7 8 9 10 11

pH

(b)


0

20

40

60

80

100

120

0 12 24 36 48 60 72 84 96 108

Rela

tive S

K ac

tivity

()

Incubation time (h)

4∘C25

∘C37

∘C

45∘C

60∘C

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120 Re

lativ

e SK

activ

ity (

)


pH 9

(b)





4 Conclusion














Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















(kDa) 2M1

66

45

35

25

18

116

14

(a)

3M(kDa)

66

45

35

25

18

116

(b)














90375mg 21789mg 4568 10336 52 226








200 400 600 800 1000 1200 1400 1600 1800

y(1)

b(4)++

b(3)

ylowast(4)y(4)

y(5)

blowast(6)

y(6)

y(7)

y(8)

y(9) y(10)

y(11)

y(12)

y(14)

y(17)


200 400 600 800 1000 1200 1400 1600

b(5)++

blowast(3)

a(7)++

y(8)++

b(8)++

blowast(9)++ b(9)++

b(10)++

y(5)

a(5)

b(4)

b(5)

y(13)++

y(6)

blowast(6)

b(14)++

y(7)

blowast(7)

b(7)

y(8)

blowast(8)

b(18)++


blowast(9)

y(10)

y(9)

blowast(10)

b(10)

y(11)

b(11)


b(12)

blowast(13)b(13)

ylowast(13)y(13)

b(28)++blowast(14)b(14)

y(14)

blowast(15)b(15)

y(15)

blowast(16)

y(3)

y(2)

alowast(4)

a(19)++


200 400 600 800 1000 1200 1400 1600 1800

ylowast(1) y(1)

blowast(2) b(4)++

y(2)

blowast(3)b(3)

b(12)

y(3)


y(9)++

blowast(6)

y(11)++

y(12)++

ylowast(13)++

y(13)++ a(14)++ y(14)++

y(7)

ylowast(8) y(8)

y(9)

y(10)

y(11)

(c) TNNTDLISEKYYVLK

200 400 600 800 1000 1200 1400 1600

b(1)

ylowast(1) y(1)a(2)

b(2)

ylowast(2) y(2)

b(3)

ylowast(3)

b(4)

ylowast(8)++

y(8)++

blowast(5) b(5)

y(5)

blowast(6) b(6)


alowast(8) a(8)

b(8)ylowast(8) y(8)

blowast(9) b(9)

ylowast(9)

ylowast(10)

(d) NLDFRDLYDPR

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+10 1009080706050403020


---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+110 200190180170160150140130120



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+210 240230220 250 300290280270260



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+310 400390380370360350340330320



---------+----410



(e)



0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Rela

tive S

K ac

tivity

()

Temperature (∘C)

(a)

Rela

tive S

K ac

tivity

()

120

100

80

60

40

20

0

5 6 7 8 9 10 11

pH

(b)


0

20

40

60

80

100

120

0 12 24 36 48 60 72 84 96 108

Rela

tive S

K ac

tivity

()

Incubation time (h)

4∘C25

∘C37

∘C

45∘C

60∘C

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120 Re

lativ

e SK

activ

ity (

)


pH 9

(b)





4 Conclusion














Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(kDa) 2M1

66

45

35

25

18

116

14

(a)

3M(kDa)

66

45

35

25

18

116

(b)














90375mg 21789mg 4568 10336 52 226








200 400 600 800 1000 1200 1400 1600 1800

y(1)

b(4)++

b(3)

ylowast(4)y(4)

y(5)

blowast(6)

y(6)

y(7)

y(8)

y(9) y(10)

y(11)

y(12)

y(14)

y(17)


200 400 600 800 1000 1200 1400 1600

b(5)++

blowast(3)

a(7)++

y(8)++

b(8)++

blowast(9)++ b(9)++

b(10)++

y(5)

a(5)

b(4)

b(5)

y(13)++

y(6)

blowast(6)

b(14)++

y(7)

blowast(7)

b(7)

y(8)

blowast(8)

b(18)++


blowast(9)

y(10)

y(9)

blowast(10)

b(10)

y(11)

b(11)


b(12)

blowast(13)b(13)

ylowast(13)y(13)

b(28)++blowast(14)b(14)

y(14)

blowast(15)b(15)

y(15)

blowast(16)

y(3)

y(2)

alowast(4)

a(19)++


200 400 600 800 1000 1200 1400 1600 1800

ylowast(1) y(1)

blowast(2) b(4)++

y(2)

blowast(3)b(3)

b(12)

y(3)


y(9)++

blowast(6)

y(11)++

y(12)++

ylowast(13)++

y(13)++ a(14)++ y(14)++

y(7)

ylowast(8) y(8)

y(9)

y(10)

y(11)

(c) TNNTDLISEKYYVLK

200 400 600 800 1000 1200 1400 1600

b(1)

ylowast(1) y(1)a(2)

b(2)

ylowast(2) y(2)

b(3)

ylowast(3)

b(4)

ylowast(8)++

y(8)++

blowast(5) b(5)

y(5)

blowast(6) b(6)


alowast(8) a(8)

b(8)ylowast(8) y(8)

blowast(9) b(9)

ylowast(9)

ylowast(10)

(d) NLDFRDLYDPR

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+10 1009080706050403020


---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+110 200190180170160150140130120



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+210 240230220 250 300290280270260



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+310 400390380370360350340330320



---------+----410



(e)



0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Rela

tive S

K ac

tivity

()

Temperature (∘C)

(a)

Rela

tive S

K ac

tivity

()

120

100

80

60

40

20

0

5 6 7 8 9 10 11

pH

(b)


0

20

40

60

80

100

120

0 12 24 36 48 60 72 84 96 108

Rela

tive S

K ac

tivity

()

Incubation time (h)

4∘C25

∘C37

∘C

45∘C

60∘C

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120 Re

lativ

e SK

activ

ity (

)


pH 9

(b)





4 Conclusion














Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




90375mg 21789mg 4568 10336 52 226








200 400 600 800 1000 1200 1400 1600 1800

y(1)

b(4)++

b(3)

ylowast(4)y(4)

y(5)

blowast(6)

y(6)

y(7)

y(8)

y(9) y(10)

y(11)

y(12)

y(14)

y(17)


200 400 600 800 1000 1200 1400 1600

b(5)++

blowast(3)

a(7)++

y(8)++

b(8)++

blowast(9)++ b(9)++

b(10)++

y(5)

a(5)

b(4)

b(5)

y(13)++

y(6)

blowast(6)

b(14)++

y(7)

blowast(7)

b(7)

y(8)

blowast(8)

b(18)++


blowast(9)

y(10)

y(9)

blowast(10)

b(10)

y(11)

b(11)


b(12)

blowast(13)b(13)

ylowast(13)y(13)

b(28)++blowast(14)b(14)

y(14)

blowast(15)b(15)

y(15)

blowast(16)

y(3)

y(2)

alowast(4)

a(19)++


200 400 600 800 1000 1200 1400 1600 1800

ylowast(1) y(1)

blowast(2) b(4)++

y(2)

blowast(3)b(3)

b(12)

y(3)


y(9)++

blowast(6)

y(11)++

y(12)++

ylowast(13)++

y(13)++ a(14)++ y(14)++

y(7)

ylowast(8) y(8)

y(9)

y(10)

y(11)

(c) TNNTDLISEKYYVLK

200 400 600 800 1000 1200 1400 1600

b(1)

ylowast(1) y(1)a(2)

b(2)

ylowast(2) y(2)

b(3)

ylowast(3)

b(4)

ylowast(8)++

y(8)++

blowast(5) b(5)

y(5)

blowast(6) b(6)


alowast(8) a(8)

b(8)ylowast(8) y(8)

blowast(9) b(9)

ylowast(9)

ylowast(10)

(d) NLDFRDLYDPR

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+10 1009080706050403020


---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+110 200190180170160150140130120



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+210 240230220 250 300290280270260



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+310 400390380370360350340330320



---------+----410



(e)



0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Rela

tive S

K ac

tivity

()

Temperature (∘C)

(a)

Rela

tive S

K ac

tivity

()

120

100

80

60

40

20

0

5 6 7 8 9 10 11

pH

(b)


0

20

40

60

80

100

120

0 12 24 36 48 60 72 84 96 108

Rela

tive S

K ac

tivity

()

Incubation time (h)

4∘C25

∘C37

∘C

45∘C

60∘C

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120 Re

lativ

e SK

activ

ity (

)


pH 9

(b)





4 Conclusion














Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


200 400 600 800 1000 1200 1400 1600 1800

y(1)

b(4)++

b(3)

ylowast(4)y(4)

y(5)

blowast(6)

y(6)

y(7)

y(8)

y(9) y(10)

y(11)

y(12)

y(14)

y(17)


200 400 600 800 1000 1200 1400 1600

b(5)++

blowast(3)

a(7)++

y(8)++

b(8)++

blowast(9)++ b(9)++

b(10)++

y(5)

a(5)

b(4)

b(5)

y(13)++

y(6)

blowast(6)

b(14)++

y(7)

blowast(7)

b(7)

y(8)

blowast(8)

b(18)++


blowast(9)

y(10)

y(9)

blowast(10)

b(10)

y(11)

b(11)


b(12)

blowast(13)b(13)

ylowast(13)y(13)

b(28)++blowast(14)b(14)

y(14)

blowast(15)b(15)

y(15)

blowast(16)

y(3)

y(2)

alowast(4)

a(19)++


200 400 600 800 1000 1200 1400 1600 1800

ylowast(1) y(1)

blowast(2) b(4)++

y(2)

blowast(3)b(3)

b(12)

y(3)


y(9)++

blowast(6)

y(11)++

y(12)++

ylowast(13)++

y(13)++ a(14)++ y(14)++

y(7)

ylowast(8) y(8)

y(9)

y(10)

y(11)

(c) TNNTDLISEKYYVLK

200 400 600 800 1000 1200 1400 1600

b(1)

ylowast(1) y(1)a(2)

b(2)

ylowast(2) y(2)

b(3)

ylowast(3)

b(4)

ylowast(8)++

y(8)++

blowast(5) b(5)

y(5)

blowast(6) b(6)


alowast(8) a(8)

b(8)ylowast(8) y(8)

blowast(9) b(9)

ylowast(9)

ylowast(10)

(d) NLDFRDLYDPR

---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+10 1009080706050403020


---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+110 200190180170160150140130120



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+210 240230220 250 300290280270260



---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+310 400390380370360350340330320



---------+----410



(e)



0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Rela

tive S

K ac

tivity

()

Temperature (∘C)

(a)

Rela

tive S

K ac

tivity

()

120

100

80

60

40

20

0

5 6 7 8 9 10 11

pH

(b)


0

20

40

60

80

100

120

0 12 24 36 48 60 72 84 96 108

Rela

tive S

K ac

tivity

()

Incubation time (h)

4∘C25

∘C37

∘C

45∘C

60∘C

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120 Re

lativ

e SK

activ

ity (

)


pH 9

(b)





4 Conclusion














Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Rela

tive S

K ac

tivity

()

Temperature (∘C)

(a)

Rela

tive S

K ac

tivity

()

120

100

80

60

40

20

0

5 6 7 8 9 10 11

pH

(b)


0

20

40

60

80

100

120

0 12 24 36 48 60 72 84 96 108

Rela

tive S

K ac

tivity

()

Incubation time (h)

4∘C25

∘C37

∘C

45∘C

60∘C

(a)

0 10 20 30 40 50 600

20

40

60

80

100

120 Re

lativ

e SK

activ

ity (

)


pH 9

(b)





4 Conclusion














Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Highly Effective Renaturation of a ...downloads.hindawi.com/journals/bmri/2014/324705.pdf · Highly Effective Renaturation of a Streptokinase from Streptococcus pyogenes