Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.
A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.
Cui
Taian
T
School of Chemical and Life Sciences, Singapore Polytechnic, 500 Dover Road, Singapore 139651, Republic of Singapore. tacui@sp.edu.sg
Gao
Yaojun
Y
Liew
Oi Wah
OW
Puah
Chum Mok
CM
Gutte
Bernd
B
D005591:Chemical Fractionation / Q000379:methods; D047169:Cyclotides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D006839:Hydrocarbons, Acyclic / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D047169:Cyclotides; D006839:Hydrocarbons, Acyclic; D010936:Plant Extracts; D011993:Recombinant Fusion Proteins; C091930:kalata B1; D013879:Thioredoxins