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Rivista di Biologia Molecolare e Biotecnologie

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Minerva Biotecnologica 1998 December;10(4):174-77

lingua: Inglese

Hydrophobic interaction chromatography ­applied to pur­ifi­ca­tion of recom­bi­nant streptokinase

Pérez N. 1, Urrutia E. 1, Camino J. 1, Orta D. R. 1, Torres Y. 1, Martinez Y. 2, Cruz M. 2, Alburquerque S. 1, Gil M. R. 1, Hernandez I. 1

1 Streptokinase Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba;
2 Quality Control Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba


Background. Recombinant streptokinase (rSk) is a strep­to­coc­cal pro­tein ­cloned in E. ­coli.11 Several meth­ods ­have ­been ­described for strep­tok­i­nase pur­ifi­ca­tion: ion ­exchange chrom­a­tog­ra­phy,12 affin­ity chrom­a­tog­ra­phy ­with ­canine plas­min13 and chrom­a­tog­ra­phy on immo­bi­lized acy­lat­ed ­human plas­mi­no­gen.14 Monoclonal anti­bod­ies ­anti-rSk immo­bi­lized to Sepharose15 ­have ­been ­used too. Recently ­this pro­tein was pur­i­fied ­using HIC.
Methods. rsk (­CIGB, Cuba) was pro­duced by fer­men­ta­tion ­strain K12 of E. ­coli,11 the pro­tein was recov­ered ­after ­washed pel­let, cel­lu­lar dis­rup­tion and sol­u­bil­iza­tion. Several pur­ifi­ca­tion ­assays ­were ­done ­using TSK-Butyl (Tosohaas, Japan) as a sup­port for hydro­pho­bic inter­ac­tion chrom­a­tog­ra­phy (HIC). The pro­tein was load­ed to the col­umn ­with 1 M of ammo­ni­um sul­fate ­before ­being ­washed ­using an elu­tion gra­di­ent ­from 0.5 to 0 M of ammo­ni­um sul­fate, in ­order to deter­mine the elu­tion ­point of the rSk.
Results. We ­could deter­mine ­that ­this pro­tein is part­ly hydro­pho­bic, ­this deter­mi­na­tion was ­shown by anal­y­sis of its ami­noacid­ic ­sequence. This pro­tein has 415 ami­noacids of ­which 36% are non ­polar. The absorp­tion capac­ity for TSK Butyl 650 S var­ies ­from 15 to 20 mg/mL. The opti­mun elu­tion ­point was ­obtained ­using 0.25 M of ammo­ni­um sul­tate, the elut­ed mate­ri­al was ­obtained ­with a ­high lev­el of pur­ity (<1% of con­tam­i­nants). The recov­ery of rSk was ­about 49% ­using the ­mean of ­five ­assays.
Conclusions. The experi­men­tal pro­cess eval­u­at­ed ­could be effi­cient­ly insert­ed in a down­stream pro­cess to ­obtain recom­bi­nant streptokinase high­ly pur­i­fied as ­final prep­ar­a­tion and ­good recov­ery.

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