Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections.

Brinkmann, Ulrich; Büchner, Johannes; Pastan, Ira

doi:10.1073/pnas.89.7.3075

Cited by 86 publications

(51 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…ARGININE was first used in refolding of human tissue type plasminogen activator [18,19]. Since then, it has been used for refolding of a variety of proteins including casein kinase II, [20], Fab antibody fragments [18,21], growth hormone [22], human gamma interferon, [23], human matrix metalloproteinase-7 human neurotrophins [24] human p53 tumor suppressor protein [25], interleukin-21 [26], interleukin-6 receptor [27], lysozyme [28], single-chain Fv fragments [29], and singlechain immunotoxins [18,30]. However, arginine may act as a protein-denaturant, which limits the expansion of its applications.…”

Section: Arginine (Arg)mentioning

confidence: 99%

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Alibolandi¹,

Mirzahosei²

2011

American J. of Biochemistry and Molecular Biology

View full text Add to dashboard Cite

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

show abstract

Section: Arginine (Arg)mentioning

confidence: 99%

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Alibolandi¹,

Mirzahosei²

2011

American J. of Biochemistry and Molecular Biology

View full text Add to dashboard Cite

show abstract

“…The final modification of the scFv-BSRNase fusion protein constructs was the inclusion of a spacer/linker sequence between the scFv and the BSRNase. This was added to separate further the two portions of the molecule to allow better independent folding (as observed for Pseudomonas exotoxin immunotoxins ;Brinkmann et al, 1992). In addition, it has been shown that the N-termini of the BSRNase dimer are exchanged across the subunit interface in some forms of the dimer .…”

Section: Enzyme-linked Immunosorbent Assays and Western Blotsmentioning

confidence: 99%

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

Deonarain

Epenetos²

1998

Br J Cancer

View full text Add to dashboard Cite

Summary Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable antitumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 1 04-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia cofi (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.

show abstract

“…It has been reported that transforming growth factor a, IL2, IL3, IL4, IL6, GM-CSF, and insulin-like growth factor I all bind with 20-to 250-fold reduced affinity after fusion to another protein (7,(13)(14)(15)(16). Previously, the options available for improving the function of a fusion protein were limited to switching the order of the two proteins, attaching linkers in between the two proteins, or mutating one of the proteins to decrease junctional effects (13,17).…”

mentioning

confidence: 99%

A circularly permuted recombinant interleukin 4 toxin with increased activity.

Kreitman

Puri

Pastan

1994

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor. With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the d. However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor. To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fria ent encoding circularly permuted interleukin 4 (H4), termed 114(38-37 In many cases, fusion of one protein to another impairs the activity of one or both proteins. This can occur if one or both proteins require one or both termini free for optimal activity. Alternatively, connection of one protein to another can impair the ability ofeither protein to properly fold. It has been reported that transforming growth factor a, IL2, IL3, IL4, IL6, GM-CSF, and insulin-like growth factor I all bind with 20-to 250-fold reduced affinity after fusion to another protein (7,(13)(14)(15)(16). Previously, the options available for improving the function of a fusion protein were limited to switching the order of the two proteins, attaching linkers in between the two proteins, or mutating one of the proteins to decrease junctional effects (13,17).We have previously reported on the properties of chimeric toxins in which IL4 was fused to recombinant forms of Pseudomonas exotoxin (PE). The fusion proteins bind to the IL4 receptor (IL4R) with only "l4% of the affinity of native IL4 (14). Several studies have indicated that the carboxyl terminus of IL4 is important for binding (18,19); therefore, it is likely that the large toxin molecule attached to the carboxyl terminus blocks the binding of 1L4 to the IL4R. We therefore developed a strategy for fusing the two proteins in which the toxin is fused to the new carboxyl terminus of circularly permuted forms of IL4. A circular permuted protein is a mutant protein in which the termini have been fused and new termini created elsewhere in the molecule. Several circularly permuted proteins have been made but none of these have been fused to other proteins (20). We reasoned that circularly permuted IL4 (CP-IL4) could be fused to a toxin so that thejunction would be in an entirely new location and allow the IL4 to bind in a more native fashion. MATERIALS AND METHODSPlasmid Construction. pRKL4EL encodes human IL4 followed by the amino acids ELKA, contains gag-ctc-gaa-gcttga (nucleotide sequences are shown by lowercase letters to avoid confusion with single-letter amino acid symbols) at the end of the coding sequence, and was derived by PCR amplification of pWDMH4 (14) and ligation into the vector pVCDT1-anti-Tac(Fv) (21). pRKL4 encodes the 129 amino acid 14 protein purified from Escherichia coli (22) and was derived from PCR amplification of pRKLAEL and ligation into the vector pVCDT1-anti-Tac(Fv). DNA encoding the toxin used for fusion to CP...

show abstract

Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: influence of interdomain connections.

Cited by 86 publications

References 32 publications

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

Retracted: Chemical Assistance in Refolding of Bacterial Inclusion Bodies

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

A circularly permuted recombinant interleukin 4 toxin with increased activity.

Contact Info

Product

Resources

About