Disulfide Bond Assignment in Human Interleukin-7 by Matrix-assisted Laser Desorption/Ionization Mass Spectroscopy and Site-directed Cysteine to Serine Mutational Analysis

Cosenza, Larry; Sweeney, E; Murphy, John R.

doi:10.1074/jbc.272.52.32995

Cited by 29 publications

(18 citation statements)

References 19 publications

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“…These phenomena have suggested that there is a conserved mechanism for receptor binding among the cytokines~Bazan, 1990a!. In this case, the solvent exposed hydrophobic surface of the IL-7 model would energetically favor protein interactions for receptor binding and might explain dimer formation seen during expression and purificatioñ Cosenza et al, 1997!. This hypothesis was tested using sitedirected Ala substitution-scanning mutagenesis along the carboxyl terminus of hIL-7.…”

Section: Discussionmentioning

confidence: 99%

Comparative model building of interleukin‐7 using interleukin‐4 as a template: A structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation

et al. 2000

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Using a combination of theoretical sequence structure recognition predictions and experimental disulfide bond assignments, a three-dimensional~3D! model of human interleukin-7~hIL-7! was constructed that predicts atypical surface chemistry in helix D that is important for receptor activation. A 3D model of hIL-7 was built using the X-ray crystal structure of interleukin-4~IL-4! as a template~Walter MR et al., 1992, J Mol Biol. 224:1075-1085 Walter MR et al., 1992, J Biol Chem 267:20371-20376!. Core secondary structures were constructed from sequences of hIL-7 predicted to form helices. The model was constructed by superimposing IL-7 helices onto the IL-4 template and connecting them together in an up-up down-down topology. The model was finished by incorporating the disulfide bond assignments Cys3, Cys142!,~Cys35, Cys130!, and~Cys48, Cys93!, which were determined by MALDI mass spectroscopy and site-directed mutagenesis~Cosenza L, Sweeney E, Murphy JR, 1997, J Biol Chem 272:32995-33000!. Quality analysis of the hIL-7 model identified poor structural features in the carboxyl terminus that, when further studied using hydrophobic moment analysis, detected an atypical structural property in helix D, which contains Cys130 and Cys142. This analysis demonstrated that helix D had a hydrophobic surface exposed to bulk solvent that accounted for the poor quality of the model, but was suggestive of a region in IL-7 that maybe important for protein interactions. Alanine~Ala! substitution scanning mutagenesis was performed to test if the predicted atypical surface chemistry of helix D in the hIL-7 model is important for receptor activation. This analysis resulted in the construction, purification, and characterization of four hIL-7 variants, hIL-7~K121A!, hIL-7~L136A!, hIL-7~K140A!, and hIL-7~W143A!, that displayed reduced or abrogated ability to stimulate a murine IL-7 dependent pre-B cell proliferation. The mutant hIL-7~W143A!, which is biologically inactive and displaces @ 125 I#-hIL-7, is the first reported IL-7R system antagonist.

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Section: Discussionmentioning

confidence: 99%

Comparative model building of interleukin‐7 using interleukin‐4 as a template: A structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation

et al. 2000

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“…In combination with enzymatic digestions, chemical modifications, and separation by high-performance liquid chromatography (HPLC), MALDI and ESI have been widely used in protein sequencing and disulfide mapping [7][8][9][10][11].…”

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confidence: 99%

Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

Zhao¹,

Almaraz

Fan

et al. 2009

J. Am. Soc. Mass Spectrom.

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Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. (J Am Soc

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“…The hIL-7 gene is located on chromosome 8q12-13, spans six exons, and has a coding region of 534 nucleotides (177 amino acids) including a signal sequence of 25 amino acids with three disulfide bonds (Cosenza et al 1997). The heterologous expression of hIL-7 protein has been tested in different hosts such as Escherichia coli (Cosenza et al 1997), Pichia pastoris (35 mg/L) (Luo et al 2009), and baculovirus (124 mg/L) (Mirzaei et al 2008).…”

Section: Introductionmentioning

confidence: 99%

“…The heterologous expression of hIL-7 protein has been tested in different hosts such as Escherichia coli (Cosenza et al 1997), Pichia pastoris (35 mg/L) (Luo et al 2009), and baculovirus (124 mg/L) (Mirzaei et al 2008). The expression level in E. coli hosts was uniformly poor in the range of 5-79 mg/L (Wickham and Walsh 2007;Zaremba-Czogalla et al 2015).…”

Section: Introductionmentioning

confidence: 99%

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture

Devi

Adivitiya

Khasa

2016

Appl Microbiol Biotechnol

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Human interleukin-7 (hIL-7) is a therapeutically important cytokine involved in lymphocyte development and survival. In previous reports, a uniformly poor expression of hIL-7 has been shown in Escherichia coli host with the problem of inclusion body formation. In this study, the role of codon optimization and N-terminus blocking using various solubility enhancer fusion tags was explored to improve its soluble expression. The use of codon optimization strategy improved its expression to 80 ± 5 mg/L at shake flask level. The utilization of pelB leader sequence resulted in an unprocessed protein in the form of cytoplasmic inclusion bodies with lower expression yields. The N-terminus fusion of small ubiquitin-like modifier (SUMO), thioredoxin (Trx), and NusA tags increased the expression in the range of 90-140 mg/L, where >90 % of the fusion protein was obtained in soluble form. The fed-batch fermentation of SUMO-tagged hIL-7 protein was optimized at bioreactor level, where a high volumetric product concentration of 2.65 g/L was achieved by controlling the plasmid segregation instability using high antibiotic concentration. The specific product yield (Y) and volumetric product concentration were 1.38 and 2.55-fold higher compared to batch results, respectively. A preparative scale affinity chromatography resulted in a high recovery yield of 50.6 mg/L with ∼90 % purity. The conformational property of purified recombinant hIL-7 from CD spectroscopy showed a typical helical structure with 31.5 % α-helix and 26.43 % β-sheet. The biological activity of purified protein was tested using IL-7-dependent murine immature B lymphocyte (2E8) cell line by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide salt (MTT) assay, where it showed a similar biological activity as standard control.

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Disulfide Bond Assignment in Human Interleukin-7 by Matrix-assisted Laser Desorption/Ionization Mass Spectroscopy and Site-directed Cysteine to Serine Mutational Analysis

Cited by 29 publications

References 19 publications

Comparative model building of interleukin‐7 using interleukin‐4 as a template: A structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation

Comparative model building of interleukin‐7 using interleukin‐4 as a template: A structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation

Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture

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