Production and Purification of Norovirus P Proteins

The P proteins of different GII.4 strains were made as described previously [19 (link)]. A cysteine-containing peptide was linked to the N (CNGRC-P) or C (P-CDCRGDCFC) terminus of the P domains to enhance P-particle formation. The cDNAs encoding the capsid P domain without the hinge were cloned into the expression vector pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ) between Bam HI and Not I sites. After sequence confirmation, the P proteins were expressed in E. coli following previously described procedures [35 (link),39 (link),40 (link)]. Briefly, the BL21 cultures were induced by IPTG (isopropyl-β-D-thiogalactopyranoside) (0.4 mM) at room temperature (22°C) overnight. The recombinant P domain-GST fusion proteins were purified using Glutathione Sepharose 4 Fast Flow resin (GE Healthcare life Sciences, NJ, USA) according to the manufacturer’s instructions. GST was removed from the P proteins by thrombin (GE Healthcare life Sciences, NJ, USA) cleavage on beads at room temperature overnight. The P-particle formation was confirmed by gel filtration, using a Superdex 200 (GE Healthcare Life-Sciences, Piscataway, NJ) size exclusion column, during which the P particles formed a peak at ~830 kDa [18 (link)].

Free full text: Click here

Dai Y.C., Zhang X.F., Xia M., Tan M., Quigley C., Lei W., Fang H., Zhong W., Lee B., Pang X., Nie J, & Jiang X. (2015). Antigenic Relatedness of Norovirus GII.4 Variants Determined by Human Challenge Sera. PLoS ONE, 10(4), e0124945.

Publication 2015

pGEX-4T-1 Glutathione Sepharose 4 Fast Flow resin thrombin Superdex 200

Capsid Cdnas Cleavage Cysteine E coli Gel filtration Glutathione Iptg P proteins Peptide Recombinant fusion proteins Resin Sepharose Strains Thrombin Vector

Corresponding Organization :

Other organizations : Cincinnati Children's Hospital Medical Center, University of Alberta, Southern Medical University

Top 5 similar protocols

Variable analysis

independent variables

The P proteins of different GII.4 strains

dependent variables

P-particle formation

control variables

The cysteine-containing peptide linked to the N (CNGRC-P) or C (P-CDCRGDCFC) terminus of the P domains to enhance P-particle formation
The cDNAs encoding the capsid P domain without the hinge were cloned into the expression vector pGEX-4T-1
The P proteins were expressed in E. coli following previously described procedures [35 (link), 39 (link), 40 (link)]
The recombinant P domain-GST fusion proteins were purified using Glutathione Sepharose 4 Fast Flow resin
GST was removed from the P proteins by thrombin (GE Healthcare life Sciences, NJ, USA) cleavage on beads at room temperature overnight

positive controls

The P-particle formation was confirmed by gel filtration, using a Superdex 200 (GE Healthcare Life-Sciences, Piscataway, NJ) size exclusion column, during which the P particles formed a peak at ~830 kDa [18 (link)]

negative controls

No negative controls were explicitly mentioned in the input protocol.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!