Simple method of cloning, overexpressing and purifying lens protein tau-crystallin

A simple method of cloning, overexpressing, and purifying eye lens protein tau-crystallin, said method comprising steps of:

• a. isolating total RNA from eye lens,
• b. synthesizing cDNA from the total RNA using a mixture of random primers and oligo dT primer in RT-PCR,
• c. designing both forward and reverse primers using synthesized cDNA to obtain PCR product,
• d. cloning the said PCR product as an insert in a plasmid,
• e. releasing the insert by double- digesting it with restriction enzymes,
• f. sub-cloning the released insert in an expression vector,
• g. transforming the sub-clone into an E.Coli expression host,
• h. inducing the host with IPTG,
• i. pelleting the induced host,
• j. re-suspending the pellet in lysis buffer for braking the cells,
• k. spinning the broken cells to obtain supernatant,
• l. loading the supernatant for gel filtration chromatography to obtain fractions,
• m. pooling the fractions that contain tau crystallin,
• n. purifying tau-crystallin from pooled fractions by ion-exchange Mono-Q FPLC,
• o. determining molecular weight and quaternary structure of the purified tau-crystallin using gel filtration superose 12 FPLC,
• p. determining the purity of tau-crystallin by SDS-polyacrylamide gel electrophoresis,
• q. determining secondary and tertiary structure by Far and Near-UV spectra respectively,
• r. obtaining cloned, overexpressed, and purified eye lens protein tau-crystallin.