Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and β (hFRβ)

Abstract The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a(+) and transformed into E. coli BL21(DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile. Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.

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d-alanine: d-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme

Biochemical Journal ◽

10.1042/bj2820747 ◽

1992 ◽

Vol 282 (3) ◽

pp. 747-752 ◽

Cited By ~ 8

Author(s):

O A M al-Bar ◽

C D O'Connor ◽

I G Giles ◽

M Akhtar

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Gene Sequence ◽

Phosphinic Acid ◽

Mature Protein ◽

Bamhi Fragment ◽

E Coli ◽

Escherichia Coli Expression ◽

Slow Binding Inhibitor

A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

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