Expression of recombinant zeta-crystallin in Escherichia coli with the help of GroEL/ES and its purification.
zeta-Crystallin is a taxon-specific crystallin found in the eye lens of guinea pig and other hystricomorph rodents and camelids. It is an NADPH:quinone oxidoreductase and is also present in low amounts in other tissues where it might act as a detoxifying enzyme. A lens-specific promoter confers lens-specific expression of the gene in high amounts where it is speculated to play a structural role in maintaining the transparency of the lens ensemble. A deletion mutation leads to autosomal dominant congenital cataract and also results in the loss of NADPH binding. In order to perform structural studies with the protein with an aim to delineate the cause of cataract in these mutant guinea pigs, recombinant zeta-crystallin was cloned and expressed in Escherichia coli. The overexpression of the protein in E. coli resulted in a major fraction of it partitioning into inclusion bodies. The co-overexpression of the bacterial chaperone system GroEL/ES along with zeta-crystallin could significantly enhance the yield of soluble protein. Active zeta-crystallin could then be purified from the E. coli using Mono Q anion exchange FPLC and was found to be identical to the native zeta-crystallin isolated from the guinea pig lens with respect to size, spectral properties, and activity.
Goenka
S
S
Centre for Cellular and Molecular Biology, Hyderabad, 500007, India.
Rao
C M
CM
D000818:Animals; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism*; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006168:Guinea Pigs; D002479:Inclusion Bodies / Q000378:metabolism; D007908:Lens, Crystalline / Q000737:chemistry; D009249:NADP / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014169:Transformation, Bacterial
D018835:Chaperonin 10; D018834:Chaperonin 60; D003459:Crystallins; D011994:Recombinant Proteins; D009249:NADP