Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein

TY - JOUR

T1 - Efficient and economic protein labeling for NMR in mammalian expression systems

T2 - Application to a preT-cell and T-cell receptor protein

AU - Mallis, Robert J.

AU - Lee, Jonathan J.

AU - Berg, Arjen Van den

AU - Brazin, Kristine N.

AU - Viennet, Thibault

AU - Zmuda, Jonathan

AU - Cross, Melissa

AU - Radeva, Denitsa

AU - Rodriguez-Mias, Ricard

AU - Villén, Judit

AU - Gelev, Vladimir

AU - Reinherz, Ellis L.

AU - Arthanari, Haribabu

N1 - Publisher Copyright: © 2024 The Protein Society.

PY - 2024/4

Y1 - 2024/4

N2 - Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the β subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 μM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

AB - Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the β subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 μM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

KW - F-labeling

KW - Expi293F

KW - glycosylation

KW - local deuteration

KW - mammalian expression

KW - methyl TROSY

KW - nuclear magnetic resonance spectroscopy (NMR)

KW - preT-cell receptor (preTCR)

KW - T-cell receptor (TCR)

UR - http://www.scopus.com/inward/record.url?scp=85188501441&partnerID=8YFLogxK

U2 - 10.1002/pro.4950

DO - 10.1002/pro.4950

M3 - Journal article

C2 - 38511503

AN - SCOPUS:85188501441

SN - 0961-8368

VL - 33

JO - Protein Science

JF - Protein Science

IS - 4

M1 - e4950

ER -