Molecular cloning and expression in Pichia pastoris of a hypoallergenic antigen 5

Vinzón, Sabrina E.; Pirpignani, María L.; Nowicki, Cristina; Bonino, Mirtha Biscoglio de Jiménez

doi:10.1016/j.pep.2010.03.029

Cited by 13 publications

(24 citation statements)

References 30 publications

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“…As shown here, this is indeed the case for the antibacterial enzyme LST. In most instances, destruction of the glycosylation sequon is accomplished by replacing the N-linked asparagine residue with glutamine, aspartic acid, serine, or alanine (40)(41)(42)(43). In the current study of LST glycosylation, all mutations at the Nlinked N125 residue led to significantly decreased enzyme activity, whereas mutations at two adjacent residues (S126 and T127) yielded fully functional aglycosylated variants.…”

Section: Discussionmentioning

confidence: 73%

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Zhao

Blazanovic

Choi

et al. 2014

Appl Environ Microbiol

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e Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A؉T/G؉C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel highlevel expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants. L ysostaphin (LST) is a glycyl-glycine zinc-dependent endopeptidase natively encoded on the pACK1 plasmid of Staphylococcus simulans (1), an environmental competitor of Staphylococcus aureus. LST is synthesized as a preproenzyme of 493 amino acids, and its pre-(36 amino acids) and pro-(211 amino acids) sequences are removed during and after secretion, respectively. Mature LST is a monomer composed of an N-terminal catalytic domain (132 amino acids), a C-terminal cell wall binding domain (102 amino acids), and a short connecting linker (13 amino acids) between the two (2). The LST enzyme selectively and efficiently degrades pentaglycine cross-links in the peptidoglycan component of S. aureus cell walls, ultimately resulting in bacterial lysis and death. Lysostaphin was discovered in the 1960s (3) and has since undergone various degrees of preclinical development and even small-scale clinical testing by different groups and organizations (4-8). Early interest in LST as a therapeutic agent waned as a result of ready access to conventional drugs, such as methicillin, but enthusiasm for LST in biomedical applications has been revived due to wide-spread antibiotic resistance and shallow antimicrobial development pipelines (9).One barrier to LST clinical applications is the high doses required to eradicate some infections. LST appeared to show good efficacy in an unresponsive leukemia patient suffering from multidrug-resistant staphylococcal pneumonia, multiple abscesses, and cellulitis (7), but this effect required a 500-mg systemic bolus of enzyme. In another study, nasal carriers o...

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Section: Discussionmentioning

confidence: 73%

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Zhao

Blazanovic

Choi

et al. 2014

Appl Environ Microbiol

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“…Recombinant Poly s 5 and Pol a 5 were produced in P. pastoris and purified as previously described [13]. A plasmid to express the recombinant Pol a 5 in P. pastoris was kindly provided by Dr. T. P. King [26].…”

Section: Methodsmentioning

confidence: 99%

“…Despite sequence similarity, preliminary studies in mice suggested that Poly s 5 constitutes a hypoallergenic variant. That is why we pursued the recombinant expression of P. scutellaris Ag 5 in the yeast Pichia pastoris , which was found to have the same immunological behavior than the purified natural protein [13]. As most of the specific antibodies in venom immunized animals are specific for discontinuous epitopes, such immunological behavior should indicate a proper folding of the recombinant protein.…”

Section: Introductionmentioning

confidence: 99%

A Naturally Occurring Hypoallergenic Variant of Vespid Antigen 5 from Polybia scutellaris Venom as a Candidate for Allergen-Specific Immunotherapy

et al. 2012

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Stings by insects from the Hymenoptera order are known to cause life-threatening allergic reactions and impair life quality. Despite the effectiveness of conventional vespid venom immunotherapy, more standardized and safer allergy vaccines are required and recombinant hypoallergenic variants are important clinical tools. Antigen 5 is a major allergen of vespid venoms and it was previously reported that Antigen 5 from Polybia scutellaris (Poly s 5) could be a hypoallergenic variant. In this work we assess the immunological behavior and allergenic activity of Poly s 5 in order to explore its suitability for specific immunotherapy. With this aim, recombinant Poly s 5 was expressed in Pichia pastoris and the presence of cross-reactive epitopes with Pol a 5, a known allergenic Antigen 5, was investigated both at the IgG and IgE levels, by ELISA assays and a basophil-mediator release assay respectively. A molecular model was also built to better understand the relationship between immunological and structural aspects. In mice, Poly s 5 induced IgG antibodies which cross-reacted with Pol a 5. However, Poly s 5 induced only minimal amounts of IgE and was a poor inducer of basophil-mediator release, even when the cells were sensitized with Pol a 5-specific IgE. Moreover, Poly s 5-specific serum showed a specific protective activity and was able to inhibit the Pol a 5-induced basophil degranulation. Structural analysis from the molecular model revealed that a few amino acid substitutions in the N-terminal region of Poly s 5 should lead to an alteration of the surface topography and electrostatic potential of the epitopes which could be responsible for its hypoallergenic behavior. These findings, taken as a whole, show that Poly s 5 is likely a naturally occurring hypoallergenic Antigen 5 variant.

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“…The use of recombinant allergens could also improve the safety profile of SIT by preventing the inclusion of these non-allergenic components present in whole venom. Furthermore, venomic analyses have allowed identification of hypoallergenic variants of venom allergens [107]. Recombinant production of hypoallergenic proteins lacking IgE-epitopes but maintaining structural determinant for T-cell recognition and induction of allergen-blocking antibodies have been suggested as a novel alternative for improvement of SIT [108].…”

Section: Polybia Paulista Venom Allergensmentioning

confidence: 99%

“…In addition to be helpful in allergy diagnosis and immunotherapy, the recombinant forms of the antigen 5 could be used in different assays for structural and functional characterization. To date, antigen 5 from venom of social Hymenoptera has been expressed in bacteria [125,126], yeast [107] and insect cells [118,127]. Some of the heterologous forms of the allergen have proved to significantly improve CRD of Hymenoptera venom allergy [105,128].…”

Section: Antigen 5 (Poly P 5)mentioning

confidence: 99%

Wasp venomic: Unravelling the toxins arsenal of Polybia paulista venom and its potential pharmaceutical applications

Pérez-Riverol

Santos-Pinto

Lasa

et al. 2017

Journal of Proteomics

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Polybia paulista (Hymenoptera: Vespidae) is a neotropical social wasp from southeast Brazil. As most social Hymenoptera, venom from P. paulista comprises a complex mixture of bioactive toxins ranging from low molecular weight compounds to peptides and proteins. Several efforts have been made to elucidate the molecular composition of the P. paulista venom. Data derived from proteomic, peptidomic and allergomic analyses has enhanced our understanding of the whole envenoming process caused by the insect sting. The combined use of bioinformatics, -omics- and molecular biology tools have allowed the identification, characterization, in vitro synthesis and recombinant expression of several wasp venom toxins. Some of these P. paulista - derived bioactive compounds have been evaluated for the rational design of antivenoms and the improvement of allergy specific diagnosis and immunotherapy. Molecular characterization of crude venom extract has enabled the description and isolation of novel toxins with potential biotechnological applications. Here, we review the different approaches that have been used to unravel the venom composition of P. paulista. We also describe the main groups of P. paulista - venom toxins currently identified and analyze their potential in the development of component-resolved diagnosis of allergy, and in the rational design of antivenoms and novel bioactive drugs.

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Molecular cloning and expression in Pichia pastoris of a hypoallergenic antigen 5

Cited by 13 publications

References 30 publications

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

A Naturally Occurring Hypoallergenic Variant of Vespid Antigen 5 from Polybia scutellaris Venom as a Candidate for Allergen-Specific Immunotherapy

Wasp venomic: Unravelling the toxins arsenal of Polybia paulista venom and its potential pharmaceutical applications

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