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A bicistronic vector backbone for rapid seamless cloning and chimerization of ??T-cell receptor sequences.

ABSTRACT: To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic ??TCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCR? and TCR? V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human ??TCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order ?-P2A-?. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.

SUBMITTER: Kropp KN

PROVIDER: S-EPMC7480877 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.

Kropp Korbinian N KN Schäufele Tim J TJ Fatho Martina M Volkmar Michael M Conradi Roland R Theobald Matthias M Wölfel Thomas T Wölfel Catherine C

PloS one 20200909 9

To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI a ...[more]

PMID: 32903281

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Project description:BackgroundTagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector generation. In conventional cloning, tags are either added on PCR primers (requiring a distinct primer and PCR product per tag) or provided on the vector (typically leaving a restriction site scar).ResultsHere we report a vector family of 40 plasmids allowing simple, seamless fusions of a single PCR product with various N- and C-terminal tags, signal sequences and promoters. The restriction site free cloning (RSFC) strategy presented in this paper relies on seamless cloning using type IIS restriction endonucleases. After cutting out a stuffer (placeholder) fragment from the vectors, a single PCR product can be directly inserted in frame into all 40 plasmids using blunt end or TA ligations, requiring only verification of the orientation. We have established a RSFC vector family for the commonly used protein expression host Pichia pastoris and demonstrated the system with the secretory expression of horseradish peroxidase (HRP). HRP fusions to four tags (Myc, FLAG, His, Strep) and two fusion proteins (GFP and MBP) showed a 31-fold difference in volumetric activities. C-terminal tagging caused in some cases almost a complete loss of function, whereas N-terminal tags showed moderate differences.ConclusionsThe RSFC vectors provide an unprecedented toolbox for expression optimization in P. pastoris. The results obtained with HRP underline the importance of comparing different tags to maximize activities of fusion proteins. In a similar fashion the RSFC strategy can be applied in other expression hosts to screen for optimal promoters, signal sequences or to facilitate the evaluation of (iso-) enzyme families.

Dataset Information

A bicistronic vector backbone for rapid seamless cloning and chimerization of ??T-cell receptor sequences.

Publications

A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.

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