Cloning and expression of pinB gene from Triticum monococum seeds.
Puroindolines (PIN) are low molecular weight, cysteine-rich, endosperm-specific, basic proteins with a unique tryptophan-rich domain found in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) as well as other members of Triticaceae. PINs appear to be involved in both flour softness as well as resistance against fungal diseases. These proteins are known to be the major components of 'friabilin' associated with the surface of water washed starch grains and possess lipid binding properties. Structural characterization of puroindolines from Triticum monococum was initiated by amplifying and subsequently cloning the corresponding pin gene into an expression vector, known as pET-32a(+). The protein contains five tryptophanin domains and ten cysteine residues. The pinB gene was fused with the 109aa Trx.Tag thioredoxin for a high-level expression. The cloning sites used for producing fusion proteins also contained cleavable His.Tag and S.tag sequences for detection and purification. After transformation of competent Origami cells, fusion protein expression was detected by growing a transformant in LB medium in the presence of 0.1 mM IPTG at room temperature for 6 hrs on a shaker. Both soluble and insoluble fusion proteins were extracted from Origami cells after sonication. Ni-NTA column (Qiagen) was used to extract and purify these fractions. Following an overnight digestion of the recombinant protein with enterokinase at room temperature, the corresponding fractions were electrophoresed in polyacrylamide gel, electroblotted onto a nitrocellulose membrane and cross-reacted with the anti-friabilin monoclonal antibody. We found that the recombinant PINB protein had a molecular weight of 16 kDa whereas TrxB was 21 kDa. Fusion protein ran at 34 kDa. PINB protein from wheat was shown to be immunologically related to a homologue, tryptophanin, in oat seed. Further study is currently underway to characterize these proteins structurally using NMR.
Mohammadi
M
M
Department of Plant Pathology and Entomology, College of Agriculture, University of Tehran, Karaj, Iran 31587-11167.
Kane
V
V
Ng
A
A
Zaidi
M A
MA
Altosaar
I
I
Ni
F
F
Tanchak
M
M
D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D008970:Molecular Weight; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012639:Seeds / Q000737:chemistry / Q000235:genetics*; D014908:Triticum / Q000737:chemistry / Q000235:genetics*
D010940:Plant Proteins; C082942:puroindoline protein, Triticum aestivum