Cytotoxicity of a recombinant ricin‐A‐chain fusion protein containing a proteolytically‐cleavable spacer sequence

FEBS Letters

Brown, Alex N.²  O'Hare, Mary¹  Hussain, Khalid¹  Thorpe, Philip E.²  Roberts, Lynne M.¹  Lord, J.Michael¹  Vitetta, Ellen S.³  Watson, Graham²  Gebhardt, Angelika¹ 

[1] Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK;Drug Targeting Laboratory, Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London WC2A 3PX, UK;Department of Microbiology and Cancer Immunobiology Centre, University of Texas, Southwestern Medical Center, Dallas, TX 75235-9048, USA

关键词: Ricin A chain;    Fusion protein;    Protein A;    Proteolytically-cleavable sequence;   

DOI  :  10.1016/0014-5793(90)81084-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide-linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti-immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.

【 授权许可】

Unknown