Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

Peraino, Jaclyn Stromp; Zhang, Huiping; Hermanrud, Christina; Li, Guoying; Sachs, David H.; Huang, Christene A.; Wang, Zhirui

doi:10.1016/j.pep.2012.01.012

Cited by 15 publications

(25 citation statements)

References 15 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: 72%

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: 72%

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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“…2) was synthesized by PCR amplification as described previously (Hermanrud et al, 2011). To construct DT390-hIL-2, the codon-optimized human IL-2 DNA was amplified using PCR primers hIL2-X1 carrying Xho I and Nco I site + hIL2 Rev carrying an Eco RI site then cloned into pwPICZalpha (Peraino et al, 2012a) between Xho I and Eco RI sites for sequencing confirmation. The insert was then cut out with Nco I + Eco RI and cloned into pwPICZalpha-DT390 (Wang et al, 2011) between Nco I and Eco RI sites yielding the final construct DT390-hIL-2 in pwPICZalpha.…”

Section: Methodsmentioning

confidence: 99%

Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

Peraino

Zhang

Rajasekera

et al. 2014

Journal of Immunological Methods

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Regulatory T cells (Treg) constitute a major inhibitory cell population which suppresses immune responses. Thus, Treg have proven to be key players in the induction of transplantation tolerance, protection from autoimmune disease and prevention of the development of effective anti-tumor immune reactions. Treg express high levels of the high affinity interleukin-2 receptor (IL-2R) consisting of IL-2Rα (CD25) together with IL-2Rβ (CD122) and the common γ-chain (CD132). An effective reagent capable of depleting Treg in vivo would facilitate better cancer treatment and allow mechanistic studies of the role of Treg in transplantation tolerance and the development of autoimmune disease. In this study, we have developed a novel bivalent human IL-2 fusion toxin along with an Ontak®-like monovalent human IL-2 fusion toxin and compared the functional ability of these reagents in vitro. Here we show that genetically linking two human IL-2 domains in tandem, thereby generating a bivalent fusion toxin, results in significantly improved capacity in targeting human CD25+ cells in vitro. Binding analysis by flow cytometry showed that the bivalent human IL-2 fusion toxin has notably increased affinity for human CD25+ cells. In vitro functional analysis demonstrated that the bivalent isoform has an increased potency of approximately 2 logs in inhibiting cellular proliferation and protein synthesis in human CD25+ cells compared to the monovalent human IL-2 fusion toxin. Additionally, we performed two inhibition assays in order to verify that the fusion toxins target the cells specifically through binding of the human IL-2 domain of the fusion toxin to the human IL-2 receptor on the cell surface. These results demonstrated that 1) both monovalent and bivalent human IL-2 fusion toxins are capable of blocking the binding of biotinylated human IL-2 to human CD25 by flow cytometry; and 2) human IL-2 blocked the fusion toxins from inhibiting protein synthesis and cellular proliferation in vitro, thus confirming that the human IL-2 fusion toxins target the cells specifically through binding to the human IL-2 receptor. We believe that the bivalent human IL-2 fusion toxin will be a more potent, and therefore, more optimal agent than the current clinically-used monovalent fusion toxin (denileukin diftitox, Ontak®) for in vivo depletion of Treg.

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“…The codon-optimized glycosylated porcine IL-2 DNA was synthesized by GenScript (Piscataway, NJ) and the codon-optimized non- N -Glycosylated porcine IL-2 DNA was generated by site-directed mutagenesis with sense PCR primer pIL2-N91A For and anti-sense PCR primer pIL2-N91A Rev (Agilent technologies). To construct DT390-pIL-2-Gly or DT390-pIL-2-Non- N -Gly, the codon-optimized glycosylated or non- N -glycosylated porcine IL-2 DNA (Figure 2) was amplified using PCR primers pIL2-X1 carrying Xho I and Nco I site + pIL2-E1 carrying an Eco RI site then cloned into pwPICZalpha (Peraino et al, 2012) between Xho I and Eco RI sites for sequencing confirmation. The insert was then cut out with Nco I + Eco RI and cloned into pwPICZalpha-DT390 (Wang et al, 2011) between Nco I and Eco RI sites yielding the final construct DT390-pIL-2-Gly or DT390-pIL-2-Non- N -Gly in pwPICZalpha.…”

Section: Methodsmentioning

confidence: 99%

“…Protein expression and purification in Pichia pastoris were performed as previously described with the following modifications (Wang et al, 2011; Peraino et al, 2012). A Ni-Sepharose fast flow resin (GE healthcare) was used for the purification.…”

Section: Methodsmentioning

confidence: 99%

“…Porcine IL-2 fusion toxins were eluted using 40 mM imidazole. Western blot analysis, FACS analysis, FACS competition/blocking analysis and K D determination were all performed as previously described (Peraino et al, 2012) using LCL13271 cells (Cho et al, 2007). The Ontak ® -like monovalent human IL-2 fusion toxin (DT390-hIL-2) used as a control for our in vitro assay was constructed, expressed and purified exactly same as the monovalent porcine IL-2 fusion toxin.…”

Section: Methodsmentioning

confidence: 99%

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Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

Peraino

Schenk

et al. 2013

Journal of Immunological Methods

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Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25+ B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins’ ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25+ cells for studying immune regulation.

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Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

Cited by 15 publications

References 15 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

Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

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