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Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.
Asunto(s)
Escherichia coli , Interleucina-2 , Proteínas Recombinantes de Fusión , Solubilidad , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interleucina-2/genética , Interleucina-2/biosíntesis , Interleucina-2/química , Interleucina-2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Expresión Génica , Cromatografía de Afinidad , Clonación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismoRESUMEN
@#Objective To optimize the expression conditions(expression and induction conditions)and purification methods(non-denaturing and denaturing purification)of recombinant Hq001 protein in salivary glands of Haemaphysalis qinghaiensis.Methods The recombinant plasmid pET-30a-Hq001 was transformed into competent cells E.coil BL21(DE3),E.coil Rosetta(DE3)and E.coil ArcticExpress(DE3)pRARE2 respectively for the selection of an optimal expression strain.The final concentration of IPTG(0,0.5,1.0 mmol/L),induction temperature(20,25 ℃)and induction time(0,2,4,6,8 h)were optimized.The recombinant bacteria expressed under the ideal induction condition were homogenized by French press and the target protein was purified by passing through a Ni-NTA affinity chromatography column under either native(denaturationrenaturation-column chromatography)or denatured conditions(denaturation-column chromatography-renaturation).The purified products were analyzed by 12% SDS-PAGE.Results E.coil BL21(DE3)was proved to be the most suitable strain for the expression of recombinant Hq001 protein.The optimum induction condition was induction with 0.5 mmol/L IPTG for 4 h at 25 ℃.The target protein with a relative molecular mass of approximately 18 800 was obtained by non-denaturing purification method,and the size was consistent with the expectation.Conclusion The recombinant protein rHq001 in salivary glands of Haemaphysalis qinghaiensis can be obtained by the optimized expression conditions and purification methods.
RESUMEN
Search for an efficacious antileishmanial vaccine has led to clinical trials of numerous vaccine candidates in the past few decades. As no promising candidate has emerged from these studies, novel vaccine modalities and vaccine assessment techniques are still emerging for antileishmanial vaccine development. Briefly, this chapter discusses: (a) history and timeline of antileishmanial vaccine development; (b) techniques utilized for developing whole-parasite and subunit-based antileishmanial vaccine formulations, and (c) immunogenicity and post-challenge protective efficacy assessment of vaccine candidates.
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Desarrollo de Vacunas , Antiprotozoarios/uso terapéutico , Vacunas de SubunidadRESUMEN
The expression and downstream purification of membrane proteins is the prerequisite for biophysical and structural studies of this major source of therapeutic targets. The gram-positive bacterium Lactococcus lactis is an attractive option for heterologous membrane protein expression and purification thanks to advantageous characteristics such as mild proteolytic activity and small genome size. Vectors designed for gene transcription under the control of inducible promoters are readily available. Specifically, the tightly regulated nisin-inducible gene expression system (NICE) allows to fine-tune the overexpression of different gene products. The expressed protein engineered with a suitable tag can be readily detected and purified from crude membrane extracts. The purpose of this protocol chapter is to detail the procedures of cloning, expression, isolation of the membrane vesicles, and affinity purification of a membrane protein of interest in L. lactis.
Asunto(s)
Clonación Molecular/métodos , Lactococcus lactis , Proteínas de la Membrana , Conformación Proteica , Proteínas Recombinantes , Membrana Celular/química , Membrana Celular/metabolismo , Fraccionamiento Químico/métodos , Cristalografía por Rayos X/métodos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Lactococcus lactis/química , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Nisina/química , Nisina/genética , Nisina/metabolismo , Organismos Modificados Genéticamente , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia/métodos , Transformación BacterianaRESUMEN
Interleukin (IL)-38 is the latest member of the IL-1 cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-38, its precise functions are poorly understood. In the present study, the cloning, expression, purification, and activity analysis of recombinant human IL-38 was described. Human IL-38 cDNA was cloned into the prokaryotic expression vector pET-44. The recombinant IL-38 containing a C-hexahistidine tag was expressed in Escherichia coli BL21 (DE3) which induced by isopropyl-ß-D-thiogalactoside. The expressed fusion protein was purified by Ni-NTA affinity chromatography. IL-38 protein was largely found in the soluble fraction. The purified IL-38 appeared a single band on SDS-PAGE, the yield of IL-38 was 4 mg from 1 L of bacterial culture, and the purity was more than 98% with low endotoxin level (<0.1 EU/µg). Western blotting confirmed the identity of the purified protein. Activity analysis showed that IL-38 can inhibit effectively the expression of proinflammatory cytokines, such as tumor necrosis factor-α, IL-1ß, IL-17, and monocyte chemoattractant protein-1 in lipopolysaccharide-activated THP-1 cells. The production and characterization of biologically active IL-38 will be beneficial for its potential role in clinical applications.
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Vectores Genéticos/metabolismo , Interleucinas/biosíntesis , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Línea Celular Tumoral , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/biosíntesis , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Histidina/genética , Histidina/metabolismo , Humanos , Interleucina-17/antagonistas & inhibidores , Interleucina-17/biosíntesis , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/biosíntesis , Interleucinas/genética , Interleucinas/aislamiento & purificación , Interleucinas/farmacología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Enteropeptidase (EC 3.4.21.9) is the glycoprotein enzyme in the small intestine that triggers the activation of the zymogens in pancreatic juice by converting trypsinogen into trypsin. Because of its physiological significance, there have been many studies on the expression, purification, and characterization of enteropeptidase from different species. The baculovirus expression system has been commonly used in research communities and scientific industries for the production of high levels of recombinant proteins, which require posttranslational modifications for functional activity. In the present study, we isolated bovine enteropeptidase catalytic subunit gene from Bos taurus indicus (GenBank accession no. KC756844), and cloned it in pFast Bac HT "A" baculovirus expression donor vector, under the polyhedrin promoter. Recombinant bovine enteropeptidase was expressed in SF-9 insect cells with high expression levels. Recombinant enteropeptidase was purified using Ni-NTA affinity chromatography. A 6-mg quantity of pure active protein was obtained from 100 mL culture using this approach. Its activity and kinetic parameters were determined by cleavage of its fluorogenic substrate Gly-(Asp) 4-Lys-ß-naphthylamide. The recombinant bovine enteropeptidase showed a K m value of 0.75 ± 0.02 mM with K cat 25 ± 1 s.