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

Abstract

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococoal protein A (PA) have been produced in E. coii. Construcb 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 specitically depurinating 28 S ribosomal RNA (via RTA). Howevtx, 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 ammo acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a dis~~de-links 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 Iinked by a d&hide bond to PA. The cleaved fusion protein was highly toxic to Saudi cells coated with ~~-~~~oglob~in antibody suiting that the RTA could be released from the PA by reduction witbin the oytosol.Ricin A chain; Fusion protein; Protein A, ~oteol~ti~ily-cl~~ble sequexe

Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins

“…A C-terminal Lys-Asp-Glu-Leu sequence (KDEL) was attached by amplifying the BSRNase gene with the C-terminal primer BSRKDEL 5'-CTGGGCGTGGAATTCTTATTACAGTTCGTC-CTTCACTGAAGCATCGAA-3' and the N-terminal primer BSRNXHO. A 22-amino-acid sequence containing the diphtheria toxin disulphide loop (O'Hare et al, 1990) was inserted between the scFv and BSRNase as a XhoVISalI cassette obtained by PCR amplification from clone pRAS50 (R Spooner, unpublished results) using the primers DTLOOPFW 5'-GGCTGACGTCTCGAGATCAAGCGCTCTAGCCTC-GAAGGTGGGTGCGCTGGTAATAG-3' and DTLOOPBK 5'-AATTCGGACGTCGACGTCGAGACCACCGCAAGACA-GGC-3'. Six constructs were made in total, with different permutations of N-terminal linker, C-terminal KDEL and diphtheria toxin disulphide loop.…”

“…The immunotoxins described in this report differ by the addition of sequences designed to improve activity and potentiate the cytotoxicity of the molecule, based upon previous work; the addition of a disulphide-bond-forming loop from diphtheria toxin can greatly increase immunotoxin cytotoxicity (O'Hare et al, 1990) and a 'KDEL' endoplasmic reticulum retention signal can increase ricin immunotoxin cytotoxicity by ten-to 250-fold (Wales et al, 1993). In addition, it has been shown that peptides linking chimaeric molecules can modulate the activity of the molecule .…”

RiVax, a recombinant ricin subunit vaccine, protects mice against ricin delivered by gavage or aerosol

“…Briefly, the gene for the enzymatically active wild type RTA, kindly provided by Dr. J. Michael Lord, Department of Biology Sciences, University of Warwick, Coventry, U.K. [ 13,14 ], was genetically altered to eliminate both its cytotoxic activity (Y80A) and its ability to induce vascular leak (V76M). This construct was then inserted into pET28a (Novagen, Madison, WI), which relies on kanamycin rather than ampicillin for selection.…”

Selective Cytotoxicity to HER2-Positive Tumor Cells by a Recombinant e23sFv-TD-tBID Protein Containing a Furin Cleavage Sequence

“…e23sFv-TD-tBID consists of an anti-HER2 scFv and the truncated form of Bid. The 10-amino acid DT translocation domain was chosen to link e23sFv and tBID because the proteolytically cleavable spacer was shown to enhance cell-killing activity (38,39). We used a prokaryotic expression system to produce biologically active e23sFv-TD-tBID, which retained the binding specificity for HER2 of the parental scFv and was rapidly internalized into HER2-positive target cells.…”