Recombinant Expression of α‐Bungarotoxin in <i>Pichia pastoris</i> Facilitates Identification of Mutant Toxins Engineered to Recognize Neuronal Nicotinic Acetylcholine Receptors

Levandoski, Mark M.; Caffery, Philip M.; Rogowski, Robert S.; Lin, Yingxin; Shi, Qing-Luo; Hawrot, Edward

doi:10.1046/j.1471-4159.2000.741279.x

Cited by 12 publications

(5 citation statements)

References 35 publications

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“…However, by using a refolding system that included the redox buffer and immobilized protein disulfide isomerase, the proteins seem to be refolded from the analysis of circular dichroism (Figure 4C). Further, 3F proteins can also be successfully produced in the native state using Pichia pastoris as the host system [38]. …”

Section: Discussionmentioning

confidence: 99%

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

et al. 2011

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BackgroundDirected evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution.ResultsWe have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function.ConclusionsThis study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.

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Section: Discussionmentioning

confidence: 99%

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

et al. 2011

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“…It has been demonstrated that the small protein toxin ␣-BGT binds specifically to the nAChR and blocks neurotransmission by acetylcholine in Drosophila (37) as in vertebrates. We used a synthetic ␣-BGT gene (15) to generate transgenic flies that could express ␣-BGT under the control of the UAS promoter. The GAL4 drivers GMR-GAL4 (17), eyeless-GAL4, and m␦-GAL4 (14) were used to express the ␣-BGT transgene in the developing visual system.…”

Section: Resultsmentioning

confidence: 99%

Nonvesicular release of acetylcholine is required for axon targeting in theDrosophilavisual system

Yang

Kunes²

2004

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

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We report evidence for a developmental role of acetylcholine in axon pathfinding in the Drosophila visual system. Acetylcholine was detected on photoreceptor axons during their navigation to target sites in the brain, a time well before the formation of functional synapses. The pattern of photoreceptor axon projections was severely disrupted when acetylcholine synthesis or metabolism was altered or eliminated, or when transgenic ␣-bungarotoxin, a nicotinic acetylcholine receptor antagonist, was expressed in the developing eye or brain. The requirement for acetylcholine signaling exists before photoreceptor neurons form synaptic connections and does not require the function of vesicular acetylcholine transporter protein. That this early effect of acetylcholine is mediated through nonvesicular release is further supported by the observation that transgenic expression of tetanus toxin, a blocker of neurotransmitter release via synaptic vesicles, did not cause similar photoreceptor axon projection defects. These observations support the notion that a form of acetylcholine secretion mediates the behavior of growth cones during axon pathfinding.I t has been generally thought that the release of neurotransmitter at axon terminals reflects synaptic activity and contributes to the development of synaptic connectivity via activitydependent processes. However, a growing body of evidence suggests that neurotransmitters function in early development in processes that can be independent of synapses or synaptic activity, such as cell proliferation, differentiation, migration, axon outgrowth, and axon branching. In these roles, neurotransmitters are apparently released by mechanisms that are distinct from the conventional synaptic vesicular pathway (1, 2). For example, it has been shown that the neurotransmitters ␥-aminobutyric acid and glutamate can be released in a Ca 2ϩ -and soluble N-ethylmaleimide-sensitive factor attachment protein receptor-independent manner, before synapse formation (3).The effects of loss of acetylcholine synthesis on branching patterns of motor axons is particularly interesting in light of the earlier observation that neurotransmitters may function as chemical signals in axon pathfinding. Experiments on isolated embryonic chick and Xenopus neurons (4, 5) and Drosophila CNS neurons (6) have indicated that the transmitter acetylcholine is synthesized very early in neural development and is present on growing axons well before they reach their target fields or establish functional synapses. Experiments in culture have supported the notion that acetylcholine may have a role in axon navigation. An acetylcholine gradient can cause a growth cone to change direction (7). These studies raise the possibility that nonsynaptic release of neurotransmitter might play a role in regulating growth cone behavior before synaptogenesis.We have undertaken an investigation of the role of small molecule neurotransmitters in the development of the Drosophila visual system. In this system, light transduction depends on synthe...

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“…), and all previous attempts included an additional refolding process [ 28 – 30 ] . Other expression systems were also attempted, such as Pichia Patoris [ 31 – 33 ] , or Eukaryotic expression systems [ 34 ]. While these attempts obtained recombinant three finger proteins (rTFP) and met the end claimed in the research, all these methods suffered from sophisticated post-purification cleavage of fusion tags and low yield.…”

Section: Introductionmentioning

confidence: 99%

Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes

Xu,

Lei,

et al. 2024

Microb Cell Fact

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Background The three-finger proteins are a collection of disulfide bond rich proteins of great biomedical interests. Scalable recombinant expression and purification of bioactive three-finger proteins is quite difficult. Results We introduce a working pipeline for expression, purification and validation of disulfide-bond rich three-finger proteins using E. coli as the expression host. With this pipeline, we have successfully obtained highly purified and bioactive recombinant α-Βungarotoxin, k-Bungarotoxin, Hannalgesin, Mambalgin-1, α-Cobratoxin, MTα, Slurp1, Pate B etc. Milligrams to hundreds of milligrams of recombinant three finger proteins were obtained within weeks in the lab. The recombinant proteins showed specificity in binding assay and six of them were crystallized and structurally validated using X-ray diffraction protein crystallography. Conclusions Our pipeline allows refolding and purifying recombinant three finger proteins under optimized conditions and can be scaled up for massive production of three finger proteins. As many three finger proteins have attractive therapeutic or research interests and due to the extremely high quality of the recombinant three finger proteins we obtained, our method provides a competitive alternative to either their native counterparts or chemically synthetic ones and should facilitate related research and applications.

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Recombinant Expression of α‐Bungarotoxin in Pichia pastoris Facilitates Identification of Mutant Toxins Engineered to Recognize Neuronal Nicotinic Acetylcholine Receptors

Cited by 12 publications

References 35 publications

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

Nonvesicular release of acetylcholine is required for axon targeting in theDrosophilavisual system

Scalable production of recombinant three-finger proteins: from inclusion bodies to high quality molecular probes

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