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A Highly Versatile Expression System for the Production of Multiply Phosphorylated Proteins.

ABSTRACT: Genetic Code Expansion (GCE) can use TAG stop codons to guide site-specific incorporation of phosphoserine (pSer) into proteins. To eliminate prematurely truncated peptides, improve yields, and enhance the production of multiphosphorylated proteins, Release Factor 1 (RF1)-deficient expression hosts were developed, yet these grew slowly and their use was associated with extensive misincorporation of natural amino acids instead of pSer. Here, we merge a healthy RF1-deficient E. coli cell line with a high-efficiency pSer GCE translation system to produce a versatile pSer GCE platform in which only trace misincorporation of natural amino acids is detected even when five phosphoserines were introduced into one protein. Approximately 400 and 200 mg of singly and doubly phosphorylated GFP per liter of culture were obtained. Importantly, the lack of truncated protein permits expression of oligomeric proteins and the use of N-terminal solubility-enhancing proteins to aid phospho-protein expression and purification. To illustrate the enhanced utility of this system, we produce doubly phosphorylated STING (Stimulator of Interferon Genes), as well as triply phosphorylated BAD (Bcl2-associated agonist of cell death) complexed with 14-3-3, in quantity, purity, and homogeneity sufficient for structural biology applications. We anticipate that the facile access to phosphoproteins enabled by this system, which we call pSer-3.1G, will expand studies of the phospho-proteome.

SUBMITTER: Zhu P

PROVIDER: S-EPMC7737670 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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A Highly Versatile Expression System for the Production of Multiply Phosphorylated Proteins.

Zhu Phillip P Gafken Philip R PR Mehl Ryan A RA Cooley Richard B RB

ACS chemical biology 20190617 7

Genetic Code Expansion (GCE) can use TAG stop codons to guide site-specific incorporation of phosphoserine (pSer) into proteins. To eliminate prematurely truncated peptides, improve yields, and enhance the production of multiphosphorylated proteins, Release Factor 1 (RF1)-deficient expression hosts were developed, yet these grew slowly and their use was associated with extensive misincorporation of natural amino acids instead of pSer. Here, we merge a healthy RF1-deficient <i>E. coli</i> cell li ...[more]

PMID: 31243963

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Project description:The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

Dataset Information

A Highly Versatile Expression System for the Production of Multiply Phosphorylated Proteins.

Publications

A Highly Versatile Expression System for the Production of Multiply Phosphorylated Proteins.

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