title: Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies.
abstract: High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.
authors.affiliation: Department of Chemical Engineering, National University of Singapore.
authors.forename: H
authors.initials: H
authors.lastname: Jin
authors.forename: M S
authors.initials: MS
authors.lastname: Uddin
authors.forename: Y L
authors.initials: YL
authors.lastname: Huang
authors.forename: W K
authors.initials: WK
authors.lastname: Teo
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D002479:Inclusion Bodies; D008233:Lymphotoxin-alpha / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins