Accurate disulfide formation in Escherichia coli: overexpression and characterization of the first domain (HF6478) of the multiple Kazal-type inhibitor LEKTI.
The human hemofiltrate peptide HF6478, a putative serine proteinase inhibitor, which is part of the precursor protein LEKTI, was cloned, overexpressed, and purified. HF6478 contains two disulfide bridges with 1-4, 2-3 connectivity, sharing partial homology to Kazal-type domains and other serine proteinase inhibitors. It was expressed as thioredoxin (Trx) fusion protein, and disulfide formation occurred in the oxidative cytoplasm of Escherichia coli Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation. The soluble fusion protein was purified using metal-chelating affinity chromatography. Cleavage of the Trx fusion protein with factor Xa and subsequent purification yielded the final product in amounts sufficient for structural studies. Characterization of recombinant HF6478 was done by amino acid sequencing, mass spectrometry, capillary zone electrophoresis, and CD spectroscopy. Taking the blood filtrate peptide HF6478 as example, we present a strategy which should facilitate the expression of different extracellular proteins in the E. coli cytoplasm.
Lauber
T
T
Lehrstuhl für Biopolymere, Universität Bayreuth, Universitätstrasse 30, 95447 Bayreuth, Germany.
Marx
U C
UC
Schulz
A
A
Kreutzmann
P
P
Rösch
P
P
Hoffmann
S
S
D002352:Carrier Proteins; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D053491:Proteinase Inhibitory Proteins, Secretory; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D004220:Disulfides; D053491:Proteinase Inhibitory Proteins, Secretory; D011993:Recombinant Fusion Proteins; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D015842:Serine Proteinase Inhibitors; D013879:Thioredoxins; D015951:Factor Xa