Purification of Recombinant Mouse C-Reactive Protein from Pichia Pastoris GS115 by Nickel Chelating Sepharose Fast-Flow Affinity Chromatography and P-Aminophenyl Phosphoryl Choline Agarose Resin Affinity Chromatography in Tandem

2021 ◽  
Author(s):  
Bin Cheng ◽  
Di Wu ◽  
Ke Wu ◽  
Xiao-Ping Huang ◽  
Jian-Min Lv ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Affinity Chromatography ◽  
Protein Function ◽  
Recombinant Expression ◽  
Expression System ◽  
Post Translational Modification ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Phosphoryl Choline ◽  
Fast Flow

Abstract C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p-Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands.

scholarly journals Optimization of the secretory expression of recombinant human C-reactive protein in Pichia pastoris

3 Biotech ◽  
2017 ◽  
Vol 7 (5) ◽  
Author(s):  
Junming Li ◽  
Chengming Sun ◽  
Lei Chen ◽  
Lihui Sun ◽  
Lijun Duan ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
C Reactive Protein ◽  
Secretory Expression ◽  
Reactive Protein

Abstract 207: C-Reactive Protein Signaling via Neurokinin B Receptor Contributes to the Pathophysiology of Preeclampsia

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Nicholas Parchim ◽  
Wei Wang ◽  
Chen Liu ◽  
Athar Siddiqui ◽  
Rodney Kellems ◽  
...  
Keyword(s):  
Placental Tissue ◽  
Protein Signaling ◽  
Post Translational Modification ◽  
C Reactive Protein ◽  
Neurokinin B ◽  
Bacterial Membranes ◽  
Reactive Protein ◽  
Nk3 Receptor ◽  
Invasion Index

Introduction: C-Reactive Protein (CRP) is an important immunomodulatory molecule. Early studies have indicated that CRP is elevated in preeclamptic patient sera, but its function remains unclear. CRP is known to bind with phosphocholine (PC) of bacteria membrane to kill bacteria by triggering innate immune response. Phosphocholination is a post-translational modification mediated by PC transferase, an enzyme reported expressed only in two tissues, including placenta and testis. Recent studies identified that Neurokinin B (NKB), a known, placenta-enriched pathogenic molecule for preeclampsia (PE), is phosphocholinated. This modification increases its stability, preferentially activates NK3 receptor (NK3R) and promotes PE features. Given CRP can bind to PC on bacterial membranes, the role that phosphocholination plays is a mystery. In light of NKB, being phosphocholinated in the endoplasmic reticulum and signals via the NK3R in the pathogenesis of PE, we feel that elevated CRP may be associated with NKB and contributes to the pathophysiology of PE via NK3R activation. Results: ELISA results show that CRP is significantly elevated in PE vs. control sera_65 ug/mL vs. 10 ug/ml; p<0.0001. Western blot analysis reveals that CRP, NKB, and NK3R are significantly increased in PE vs. control placental tissue. Immunohistochemistry studies indicate that all of these three molecules are abundant in syncytiotrophoblast cells. Co-immunoprecipitation demonstrates an interaction between CRP and NKB. In vitro , we provide the first evidence that CRP decreased the invasion of trophoblast cells from 0.7 ± 0.06 to 0.06 ± 0.001 invasion index; p<0.005, while treatment with SB222200 ameliorates shallow invasion from 0.28 ± 0.09 to 0.55 ± 0.07 invasion index; p<0.05. Significantly, infusion of CRP into pregnant mice induces hypertension (169.152 ± 14.7 mmHg vs. 141.916 ± 6.93 mmHg; p<0.05) and proteinuria (28.78 ± 13.56 mg albumin/μg Cr vs. 13.87 ± 3.277 mg albumin/μg Cr; p<0.05). Conclusion: Our findings demonstrate elevated CRP contributes to PE and NKB/NK3R is a new mechanism underlying CRP-mediated shallow invasion and disease development. These studies suggest novel pathogenic biomarkers and innovative therapies for PE.

scholarly journals Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

1999 ◽  
Vol 22 (3) ◽  
pp. 273-283 ◽  
Author(s):  
C Sen Gupta ◽  
RR Dighe
Keyword(s):  
Pichia Pastoris ◽  
Structure Function ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Recombinant Expression ◽  
Expression System ◽  
Biologically Active ◽  
Maximal Response ◽  
Glycoprotein Hormone

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

scholarly journals Constitutive Optimized Production of Streptokinase inSaccharomyces cerevisiaeUtilizing Glyceraldehyde 3-Phosphate Dehydrogenase Promoter ofPichia pastoris

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Ravi N. Vellanki ◽  
Ravichandra Potumarthi ◽  
Kiran K. Doddapaneni ◽  
Naveen Anubrolu ◽  
Lakshmi N. Mangamoori
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Expression Vector ◽  
Recombinant Expression ◽  
Expression System ◽  
Nitrogen Sources ◽  
Clot Lysis ◽  
Process Conditions ◽  
Scale Production ◽  
Activation Assay

A novel expression vector constructed from genes ofPichia pastoriswas applied for heterologous gene expression inSaccharomyces cerevisiae. Recombinant streptokinase (SK) was synthesized by cloning the region encoding mature SK under the control of glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter ofPichia pastorisinSaccharomyces cerevisiae. SK was intracellularly expressed constitutively, as evidenced by lyticase-nitroanilide and caseinolytic assays. The functional activity was confirmed by plasminogen activation assay andin vitroclot lysis assay. Stability and absence of toxicity to the host with the recombinant expression vector as evidenced by southern analysis and growth profile indicate the application of this expression system for large-scale production of SK. Two-stage statistical approach, Plackett-Burman (PB) design and response surface methodology (RSM) was used for SK production medium optimization. In the first stage, carbon and organic nitrogen sources were qualitatively screened by PB design and in the second stage there was quantitative optimization of four process variables, yeast extract, dextrose, pH, and temperature, by RSM. PB design resulted in dextrose and peptone as best carbon and nitrogen sources for SK production. RSM method, proved as an efficient technique for optimizing process conditions which resulted in 110% increase in SK production, 2352 IU/mL, than for unoptimized conditions.

Can C-Reactive Protein Function as a Surrogate Marker of Preeclampsia?

2002 ◽  
Vol 99 (Supplement) ◽  
pp. 14S
Author(s):  
Deena S. Tajran ◽  
Harrihar A. Pershadsingh
Keyword(s):  
Protein Function ◽  
Surrogate Marker ◽  
C Reactive Protein ◽  
Reactive Protein

scholarly journals INTERACTIONS OF C-REACTIVE PROTEIN WITH THE COMPLEMENT SYSTEM

1974 ◽  
Vol 140 (3) ◽  
pp. 631-647 ◽  
Author(s):  
Joan Siegel ◽  
Rosemarie Rent ◽  
Henry Gewurz
Keyword(s):  
Normal Serum ◽  
Protamine Sulfate ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Phosphoryl Choline ◽  
Heat Stable ◽  
Health And Disease ◽  
Serum Crp ◽  
The Complement System

Protamine sulfate was found to consume large amounts of C selectively during preincubation with sera of individuals in the "acute phase". Marked depletion of C1, C4, and C2 with minimal, if any, depletion of C3-9, was observed. The consumption was time and temperature dependent, occurring most rapidly and extensively at 37°C, 0.10 M relative salt concentration and pH 7.5–8.0; it required calcium ions. It was mediated by a heat-stable nondialyzable factor which separated with C-reactive protein (CRP) during fractionation and purification, correlated with serum CRP levels, and, like other known reactivities of CRP, was inhibited by phosphoryl choline. Preparations of CRP purified either from serum or ascites resulted in consumption of large amounts of C1, C4, and C2 when preincubated with normal serum and protamine. We conclude that CRP is a potent activator of the C system at the level of C1, and that polycations such as protamine sulfate are substrates of CRP which can bring about this activation. It seems not unlikely that one role of CRP in health and disease involves its ability to interact with the C system.

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