title: Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli.
abstract: Bacille Calmette-Guerin (BCG)-derived heat shock protein 65 (HSP65) has been demonstrated capable of assisting a fused peptide to generate the peptide-specific cellular immunity. Various HSP65 fusion proteins have been developed as therapeutic cancer vaccines. Purifying a recombinant HSP65 fusion protein with no purification tags for human use is routinely a challenge. Here, we report a scheme for purifying a non-tagged recombinant HSP65-Her2 peptide fusion protein (HSP65-Her2) from Escherichia coli. The HSP65-Her2 is being developed as an immunotherapeutic for the treatment of Her2-positive tumors. After fermentation in a 10-L fermentor, the HSP65-Her2 expressing E. coli were harvested and lysed by sonication. The recombinant HSP65-Her2 was then purified with four successive steps including Butyl-Sepharose FF, DEAE-Sepharose FF, 1% Triton X-114 phase separation and Sephadex G-25. Results showed that HSP65-Her2 was purified up to 97% purity and was able to generate Her2-specific cytotoxic T lymphocytes (CTLs), suggesting that the scheme is efficient for purifying the non-tagged HSP65-Her2 fusion protein with biological activity.
authors.affiliation: Department of Molecular Biology, Basic Medical College of Jilin University, Changchun 130021, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Feng
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.forename: Zemin
authors.initials: Z
authors.lastname: Xiang
authors.forename: Hongfei
authors.initials: H
authors.lastname: Wei
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Hu
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Dai
authors.forename: Mingli
authors.initials: M
authors.lastname: Fang
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Zhang
authors.forename: Yongli
authors.initials: Y
authors.lastname: Yu
authors.forename: Liying
authors.initials: L
authors.lastname: Wang
mesh_terms: D051940:B7-2 Antigen / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D018734:Genes, erbB-2; D006801:Humans; D066298:In Vitro Techniques; D009163:Mycobacterium bovis / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D051940:B7-2 Antigen; D001426:Bacterial Proteins; D018834:Chaperonin 60; D018833:Chaperonins; D017931:DNA Primers; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C497964:CD86 protein, human; C074119:heat-shock protein 65, Mycobacterium