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. 1997 Nov 15;328(Pt 1):159–163. doi: 10.1042/bj3280159

Expression, purification, and characterization of recombinant human glutamine synthetase.

C D Listrom 1, H Morizono 1, B S Rajagopal 1, M T McCann 1, M Tuchman 1, N M Allewell 1
PMCID: PMC1218900  PMID: 9359847

Abstract

A bacterial expression system has been engineered for human glutamine synthetase (EC 6.3.1.2) that produces approximately 60 mg of enzyme (20% of the bacterial soluble protein) and yields approx. 8 mg of purified enzyme per litre of culture. The recombinant enzyme was purified 5-fold to apparent homogeneity and characterized. It has a subunit molecular mass of approx. 45000 Da. The Vmax value obtained using a radioactive assay with ammonia and l-[G-3H]glutamic acid as substrates was 15.9 micromol/min per mg, 40% higher than that obtained in the colorimetric assay (9.9 micromol/min per mg) with hydroxylamine replacing ammonia as a substrate. Km values for glutamate were 3.0 mM and 3.5 mM, and for ATP they were 2.0 mM and 2. 9 mM for the radioactive and spectrophotometric assays respectively. The Km for ammonia in the radioactive assay was 0.15 mM. The midpoint of thermal inactivation was 49.7 degrees C. Hydroxylamine, Mg(II) and Mg(II)-ATP stabilized the enzyme against thermal inactivation, whereas ATP promoted inactivation. The pure enzyme is stable for several months in storage and provides a source for additional studies, including X-ray crystallography.

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Selected References

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