PermaPhos Kit
(Kit # 1000000226 )

Depositing Labs:   Ryan Mehl, Richard Cooley

Provided in this kit are reagents for expressing recombinant proteins with site-specific, non-hydrolyzable phosphoserine (nhpSer) in an engineered self-sufficient E. coli genetic code expansion (GCE) expression system.

This kit will be sent as individual bacterial stabs.

Original Publication

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ. Zhu P, Stanisheuski S, Franklin R, Vogel A, Vesely CH, Reardon P, Sluchanko NN, Beckman JS, Karplus PA, Mehl RA, Cooley RB. ACS Cent Sci. 2023 Apr 10;9(4):816-835. doi: 10.1021/acscentsci.3c00191. PubMed Article

Description

This kit contains reagents for using the “PermaPhos” system to express recombinant proteins that contain one or more site-specific, non-hydrolyzable phosphoserine (nhpSer) residues in E. coli. The nhpSer amino acid contains a phosphonate group, with the γ-oxygen of phosphoserine replaced by a bridging methylene (CH₂) group. nhpSer is a faithful phosphoserine mimic, having been documented to maintain the functionality of phosphoserine in many cases even when aspartate and glutamate phospho-mimetics do not.

This autonomous “PermaPhos” expression system uses an E. coli strain engineered to make very little phosphoserine, along with a plasmid that endows E. coli with a biosynthetic pathway that converts phosphoenolpyruvate into the nhpSer amino acid. Use of the biosynthetic pathway improves bioavailability of nhpSer inside the cells compared to supplementing the media with exogenous nhpSer amino acid, and this greatly improves nhpSer-protein expression levels – approximately 100 mg of super-folder GFP with encoded nhpSer per liter of culture is readily achievable, with >95% accuracy. Because the nhpSer amino acid is biosynthesized from a central metabolite, expressions are easily scaled.

This kit includes four plasmids and the engineered E. coli strain expression host: (i) pCDF-Frb-v1 expresses five enzymes to biosynthesize nhpSer; (ii) pERM2-nhpSer expresses components for installing the nhpSer amino acid into proteins during translation at TAG amber stop codons; (iii) pRBC-sfGFP WT is a control protein plasmid that expresses the wild-type fluorescent reporter protein super-folder GFP; (iv) pRBC-sfGFP-150TAG has the fluorescent reporter protein super-folder GFP gene with a TAG codon at site N150 to encode nhpSer at that residue; and (v) the E. coli expression host BL21(DE3) ΔserC strain, which is used because it makes very low levels of the native phosphoserine amino acid, which would otherwise compete with nhpSer.

Along with the kit, a detailed protocol for expressing nhpSer-containing proteins with PermaPhos, as well as strategies for confirming accurate encoding, is available. As noted in the protocol, users should first use the sfGFP control plasmids to validate that the PermaPhos expression system is working correctly in their labs. Once the system is working, and homogenous encoding of nhpSer into sfGFP-150TAG is validated, users can move on to attempt expressing their target proteins of interest by using standard cloning procedures to replace the sfGFP gene in the pRBC plasmid with that of the target protein of interest.

• Figure 1. Expression of permanently phosphorylated proteins in E. coli using PermaPhos. To express proteins with non-hydrolyzable phosphoserine (nhpSer), intracellular phosphoserine is eliminated by using a BL21(DE3) ΔserC mutant expression host in combination with overexpression of SerB (top panel), while a biosynthetic pathway consisting of FrbABCDE proteins is employed to convert the central metabolite phosphoenolpyruvate into nhpSer (bottom panel). A phosphoserine amino-acyl tRNA synthetase (Sep-RS, in blue) amino-acylates its cognate tRNA (Sep-tRNA_CUA, in yellow) with nhpSer (orange). The nhpSer is then incorporated into growing peptide chains during translation in response to the UAG/amber stop codon (middle panel). The resulting target protein containing site-specifically encoded nhpSer (middle panel, right) can be purified and used for downstream structural and functional studies, such as pulldowns from cell lysates that are rich in phosphatases in order to detect novel phosphorylation-dependent protein-protein interactions.

Protocol

Zhu P, Mehl RA, Cooley RB. (2023). Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia coli. Bio-protocol 13(21): e4861. doi: 10.21769/BioProtoc.4861 .

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The PermaPhos Kit was a gift from Ryan Mehl (Addgene kit #1000000226).”

For your Reference section:

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ. Zhu P, Stanisheuski S, Franklin R, Vogel A, Vesely CH, Reardon P, Sluchanko NN, Beckman JS, Karplus PA, Mehl RA, Cooley RB. ACS Cent Sci. 2023 Apr 10;9(4):816-835. doi: 10.1021/acscentsci.3c00191. PubMed Article

The PermaPhos Kit contains four plasmids and one bacterial strain. Please refer to the individual material pages below for more details on each item in this kit:

• pERM2-nhpSer
• pCDF-Frb-v1
• pRBC-sfGFP wild type
• pRBC-sfGFP-150TAG
• BL21(DE3) ΔserC