Recombinant Protein Production of CPXV and VACV

Full length CPXV_calpox derived recombinant A27 (rA27) was produced as described previously [38 (link)]. Extracellular domains of VACV_NYCBOH derived D8Δ (aa 2–260), H3Δ (aa 21–270) and L1Δ (aa 1–175) were custom-made by Genexpress (Berlin, Germany). Briefly, the following primers and restriction enzymes (restriction sites underlined) were used for cloning: L1ΔF (NdeI) CGTCGGCATATGGGTGCCGCGGCAAG, L1ΔR (NsiI) CCTGTACATGCATTTGTTTAGGTGCTATTT, D8ΔF (NdeI) CGTCGGCATATGCCGCAACAACTATCTCCT, D8ΔR (NsiI) CCGACGATGCATCTCTCTCAAATCGGACAACCATC, H3ΔF (NdeI) CGTCGGCATATGACATTTCCTAATGTTCAT and H3ΔR (BamHI) CGTCGGGGATCCTTATCCTGGATAACGTTTAG. Expression was induced with 2 mM isopropyl β-D-1-thiogalactopyranoside for 3 h at 37°C, His-tagged proteins were isolated under native (D8Δ) or denaturing (all other proteins) conditions and purified using Protino® Ni-IDA columns (Macherey-Nagel, Dueren, Germany) according to standard procedures. E. coli lysate was produced as previously described [39 (link)].

Free full text: Click here

Stern D., Pauly D., Zydek M., Miller L., Piesker J., Laue M., Lisdat F., Dorner M.B., Dorner B.G, & Nitsche A. (2016). Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening. PLoS ONE, 11(3), e0150110.

Publication 2016

Protino® Ni-IDA columns

E coli Primers Proteins Restriction enzymes

Corresponding Organization : Technical University of Applied Sciences Wildau

Top 5 similar protocols

Variable analysis

independent variables

Production of Full length CPXV_calpox derived recombinant A27 (rA27)
Expression of extracellular domains of VACV_NYCBOH derived D8Δ (aa 2–260), H3Δ (aa 21–270) and L1Δ (aa 1–175)

dependent variables

Not explicitly mentioned

control variables

Induction of expression with 2 mM isopropyl β-D-1-thiogalactopyranoside for 3 h at 37°C
Purification of His-tagged proteins under native (D8Δ) or denaturing (all other proteins) conditions using Protino® Ni-IDA columns

positive controls

E. coli lysate produced as previously described [39 (link)]

negative controls

Not explicitly mentioned

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!