Expression and purification of recombinant alpha-defensins and alpha-defensin precursors in Escherichia coli.
Recombinant expression of alpha-defensins can be obtained at efficient levels in Escherichia coli. Amplified alpha-defensin or pro-alpha-defensin coding cDNA sequences are cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells. Cells growing exponentially in nutrient-rich liquid medium are induced to express the recombinant protein by addition of 50 mM isopropyl beta-D-1-thiogalactopyranoside for 3-6 h. After bacterial cells collected by centrifugation are lysed in 6 M guanidine-HCl under non-reducing conditions, the expressed defensin fused to its 6xHis-34 amino acid N-terminal fusion partner is purified by affinity chromatography on nickel-NTA columns. A Met codon introduced at the N terminus of expressed Met-free peptides provides a unique CNBr cleavage site, enabling release of the alpha-defensin free of ancillary residues by sequential C18 RP-HPLC. Molecular masses of C18 RP-HPLC purified peptides are confirmed by MALDI-TOF mass spectrometry, and peptide homogeneity is assessed using analytical RP-HPLC and acid-urea polyacrylamide gel electrophoresis. alpha-Defensins prepared in this manner are biochemically equivalent to the natural molecules.
Figueredo
Sharel
S
Department of Pathology and Laboratory Medicine, School of Medicine, College of Health Sciences, University of California, Irvine, CA, USA.
Mastroianni
Jennifer R
JR
Tai
Kenneth P
KP
Ouellette
André J
AJ
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D023084:alpha-Defensins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D023084:alpha-Defensins