An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris

Degani, Genny; Colzani, Mara; Tettamanzi, Alberto; Sorrentino, Luca; Aliverti, Alessandro; Fritz, Günter; Aldini, Giancarlo; Popolo, Laura

doi:10.1016/j.pep.2015.06.012

Cited by 8 publications

(20 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The adoption of a mixed ethanol/methanol induction protocol, described in Materials and methods, increased approximately 6 times the yield of secreted VC1-His-Strep compared to the previously protocol based on methanol alone [20]. At 72 h from the induction, VC1-His-Strep accumulated in the medium as two major polypeptide bands and the yield was about 45 mg liter −1 (Fig.…”

Section: Resultsmentioning

confidence: 90%

“…We expressed a recombinant form of VC1, named VC1-His-Strep, in P. pastoris that previously proved to be a suitable host for the expression and secretion of stable and soluble forms of VC1 domain from human RAGE [20]. The C-terminal end of VC1 was fused to a 6 x Histidine (His) tag and a Strep tag (AWRHPQFGG), separated by an internal spacer.…”

Section: Resultsmentioning

confidence: 99%

“…The in-frame fusion and the absence of random mutations in the insert were verified by DNA sequencing (BMR Genomics, Padova, Italy). HIL-S1-VC1-His-Strep plasmid, linearized with Sal I, was transformed into KM71 P. pastoris cells using the “EasyComp” chemical transformation method (Invitrogen) as previously described [20].…”

Section: Methodsmentioning

confidence: 99%

“…We have shown in a previous report that VC1 can be produced as an autonomous domain in the methylotrophic yeast Pichia pastoris [20]. The glycosylation of VC1 and its secretion into the culture medium confers high solubility and stability to the protein, especially if compared to the protein expressed intracellularly in E. coli [20].…”

Section: Introductionmentioning

confidence: 92%

“…Although the recombinant V domain can be expressed in bacteria, purified and refolded in vitro and is the binding site for some ligands of RAGE, biochemical studies revealed that V and C1 form together a single integrated domain (VC1) that is the binding site for the majority of RAGE ligands [17], [18], [19]. We have shown in a previous report that VC1 can be produced as an autonomous domain in the methylotrophic yeast Pichia pastoris [20]. The glycosylation of VC1 and its secretion into the culture medium confers high solubility and stability to the protein, especially if compared to the protein expressed intracellularly in E. coli [20].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE)

et al. 2017

Self Cite

View full text Add to dashboard Cite

The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction.

show abstract

Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE)

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

Unveiling the molecular mechanisms underpinning biorecognition of early-glycated human serum albumin and receptor for advanced glycation end products

et al. 2020

View full text Add to dashboard Cite

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris

et al. 2019

Self Cite

View full text Add to dashboard Cite

Advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs), resulting from non-enzymatic modifications of proteins, are potentially harmful to human health. They directly act on proteins, affecting structure and function, or through receptor-mediated mechanisms. RAGE, a type I transmembrane glycoprotein, was identified as a receptor for AGEs. RAGE is involved in chronic inflammation, oxidative stress-based diseases and ageing. The majority of RAGE ligands bind to the VC1 domain. This domain was successfully expressed and secreted by Pichia pastoris. Out of two N-glycosylation sites, one (Asn25) was fully occupied while the other (Asn81) was under-glycosylated, generating two VC1 variants, named p36 and p34. Analysis of N-glycans and of their influence on VC1 properties were here investigated. The highly sensitive procainamide labeling method coupled to ES-MS was used for N-glycan profiling. N-glycans released from VC1 ranged from Man 9 GlcNAc 2-to Man 15 GlcNAc 2-with major Man 10 GlcNAc 2-and Man 11 GlcNAc 2-species for p36 and p34, respectively. Circular dichroism spectra indicated that VC1 maintains the same conformation also after removal of N-glycans. Thermal denaturation curves showed that the carbohydrate moiety has a small stabilizing effect on VC1 protein conformation. The removal of the glycan moiety did not affect the binding of VC1 to sugar-derived AGE-or malondialdehyde-derived ALE-human serum albumin. Given the crucial role of RAGE in human pathologies, the features of VC1 from P. pastoris will prove useful in designing strategies for the enrichment of AGEs/ALEs from plasma, urine or tissues, and in characterizing the nature of the interaction.

show abstract

An improved expression system for the VC1 ligand binding domain of the receptor for advanced glycation end products in Pichia pastoris

Cited by 8 publications

References 34 publications

A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE)

A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE)

Unveiling the molecular mechanisms underpinning biorecognition of early-glycated human serum albumin and receptor for advanced glycation end products

Insights into the effects of N-glycosylation on the characteristics of the VC1 domain of the human receptor for advanced glycation end products (RAGE) secreted by Pichia pastoris

Contact Info

Product

Resources

About