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Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila

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Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila. / Varshney, N.K.; Ramasamy, S.; Suresh, C.G.; Brannigan, J.A.; Wilkinson, A.J.

In: Acta Crystallographica Section F: Structural Biology and Crystallization Communications, Vol. 69, No. 8, 01.08.2013, p. 925-929.

Research output: Contribution to journalArticlepeer-review

Harvard

Varshney, NK, Ramasamy, S, Suresh, CG, Brannigan, JA & Wilkinson, AJ 2013, 'Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila', Acta Crystallographica Section F: Structural Biology and Crystallization Communications, vol. 69, no. 8, pp. 925-929. https://doi.org/10.1107/S174430911301943X

APA

Varshney, N. K., Ramasamy, S., Suresh, C. G., Brannigan, J. A., & Wilkinson, A. J. (2013). Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 69(8), 925-929. https://doi.org/10.1107/S174430911301943X

Vancouver

Varshney NK, Ramasamy S, Suresh CG, Brannigan JA, Wilkinson AJ. Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2013 Aug 1;69(8):925-929. https://doi.org/10.1107/S174430911301943X

Author

Varshney, N.K. ; Ramasamy, S. ; Suresh, C.G. ; Brannigan, J.A. ; Wilkinson, A.J. / Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila. In: Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 2013 ; Vol. 69, No. 8. pp. 925-929.

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@article{1b40f0b4369c4c40886855f1405175d4,
title = "Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila",
abstract = "Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of and β chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1{\AA}, = 104.1, β = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2{\AA}, β = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.",
author = "N.K. Varshney and S. Ramasamy and C.G. Suresh and J.A. Brannigan and A.J. Wilkinson",
year = "2013",
month = aug,
day = "1",
doi = "10.1107/S174430911301943X",
language = "English",
volume = "69",
pages = "925--929",
journal = "Acta Crystallographica Section F: Structural Biology and Crystallization Communications",
issn = "1744-3091",
publisher = "Wiley-Blackwell",
number = "8",

}

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TY - JOUR

T1 - Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila

AU - Varshney, N.K.

AU - Ramasamy, S.

AU - Suresh, C.G.

AU - Brannigan, J.A.

AU - Wilkinson, A.J.

PY - 2013/8/1

Y1 - 2013/8/1

N2 - Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of and β chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1Å, = 104.1, β = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2Å, β = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.

AB - Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of and β chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1Å, = 104.1, β = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2Å, β = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.

UR - http://www.scopus.com/inward/record.url?scp=84881176267&partnerID=8YFLogxK

U2 - 10.1107/S174430911301943X

DO - 10.1107/S174430911301943X

M3 - Article

AN - SCOPUS:84881176267

VL - 69

SP - 925

EP - 929

JO - Acta Crystallographica Section F: Structural Biology and Crystallization Communications

JF - Acta Crystallographica Section F: Structural Biology and Crystallization Communications

SN - 1744-3091

IS - 8

ER -