ORIGINAL ARTICLES   

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

Copyright © 1998 EDIZIONI MINERVA MEDICA

language: English

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.