Expression of mammalian liver glycolytic/ gluconeogenic enzymes in Escherichia coli

Recovery of active enzyme is strain and temperature dependent

K. Lin, Irwin J. Kurland, L. Z. Xu, A. J. Lange, J. Pilkis, M. R. El-Maghrabi, S. J. Pilkis

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase in escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase, fructose2,6-bisphosphatase, glucokinase, and fructose- 1,6-bis-phosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37°C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22°C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thin-β-d-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose- 1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37°C and was essentially unchanged at an induction temperature of 22°C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37°C. Temperature stability of the protein may also influence the recovery of active enzyme.

Original languageEnglish (US)
Pages (from-to)169-176
Number of pages8
JournalProtein Expression and Purification
Volume1
Issue number2
DOIs
StatePublished - 1990
Externally publishedYes

Fingerprint

Liver
Escherichia coli
Recovery
Temperature
Enzymes
Fructose-Bisphosphatase
Proteins
Phosphofructokinase-2
N-Acetylmuramoyl-L-alanine Amidase
Enhanced recovery
Glucokinase
Protein Stability
Fructose
Galactose
Phosphoric Monoester Hydrolases
Cytosol
Rats
Plasmids
Phosphotransferases
Hot Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression of mammalian liver glycolytic/ gluconeogenic enzymes in Escherichia coli : Recovery of active enzyme is strain and temperature dependent. / Lin, K.; Kurland, Irwin J.; Xu, L. Z.; Lange, A. J.; Pilkis, J.; El-Maghrabi, M. R.; Pilkis, S. J.

In: Protein Expression and Purification, Vol. 1, No. 2, 1990, p. 169-176.

Research output: Contribution to journalArticle

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abstract = "A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase in escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase, fructose2,6-bisphosphatase, glucokinase, and fructose- 1,6-bis-phosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37°C, but only about 1-2{\%} of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22°C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thin-β-d-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose- 1,6-bisphosphatase, a heat-stable enzyme, was 66{\%} at 37°C and was essentially unchanged at an induction temperature of 22°C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37°C. Temperature stability of the protein may also influence the recovery of active enzyme.",
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