Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin.
The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.
Benitez-Cardoza
Claudia G
CG
Programa Institucional en Biomedicina Molecular ENMyH-IPN, Guillermo Massieu Helguera No. 239 Fraccionamiento La Escalera Ticoman CP 07320, México DF, Mexico. beni1972uk@yahoo.com.mx
Rogniaux
Hélène
H
Popineau
Yves
Y
Guéguen
Jacques
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli; D015870:Gene Expression; D005903:Gliadin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D005903:Gliadin