April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
A Novel Protocol for Consistent and Enhanced Expression of Unmodified Heterologous CYP1B1 for Functional Genomics Studies in Glaucoma
Author Affiliations & Notes
  • Nasreen Ahmed
    Ophthalmology, All India Institute of Medical Sciences, New Delhi, India
  • Reetika Sharma
    Ophthalmology, All India Institute of Medical Sciences, New Delhi, India
  • Muneeb Faiq
    Department of Anatomy,Laboratory for Molecular Reproduction and Genetics, All India Institute of Medical Sciences, New Delhi, India
  • Rima Dada
    Department of Anatomy,Laboratory for Molecular Reproduction and Genetics, All India Institute of Medical Sciences, New Delhi, India
  • Daman Saluja
    Medical Biotechnology Laboratory,Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India
  • Tanuj Dada
    Ophthalmology, All India Institute of Medical Sciences, New Delhi, India
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3817. doi:
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      Nasreen Ahmed, Reetika Sharma, Muneeb Faiq, Rima Dada, Daman Saluja, Tanuj Dada; A Novel Protocol for Consistent and Enhanced Expression of Unmodified Heterologous CYP1B1 for Functional Genomics Studies in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3817.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Mutations in CYP1B1 gene cause PCG and also POAG.Heterologous expression of CYP1B1 (mutants and wild type) in bacteria is important for functional genomics. But heterologous CYP1B1 expression is difficult so investigators use modified bovine CYP17 N-terminus to improve CYP expression. This changes original protein and renders functional studies invalid.In our study we developed a novel protocol for heterologous CYP1B1 expression by examining the role of conditions and reagents for consistent expression of unmodified human CYP1B1.It yields an expression of more than 250 nmol/l of unmodified CYP1B1.

Methods: CYP1B1 expression was studied with Cloning vectors : pET28a(+), pTRC-His, pCWori(+) , pGEX-5X-3;Bacterial strains: DH5α, Codon plus, BL21, DE3, C100, Pril; Culture media: Luria-Bertani broth, peptone based terrific broth (TB) containing 1.2g peptone,2.4g yeast extract,0.4ml glycerol in 100ml H2O, tryptone based TB, peptone/tryptone mixture based TB; Time: 1.5, 2.5, 3.5, 4.5, 5.5, 12, 24, 36 hours;IPTG conc: 0.2mM to 1mM;Temp: 37°C, 30°C, 18°C;Rotations per minutes (RPM): 200, 180, 150; Heme-precursors: Thiamine hydrochloride (0.5 to 1.5mM), 5-amino levulenic acid (ALA) (0.2 to1.0mM);Trace elements: 5g Citric acid.1H2O, 5g ZnSO4.7H2O,1g Fe(NH4)2(SO4)2.6H2O,0.25g CuSO4.5H2O,0.05g MnSO4.1H2O, 0.05g H3BO3,0.05g Na2MoO4.2H2O dissolved in100ml H2O. Use of Phosphate buffer system against without buffering. CYP1B1 expression was detected using western blot and concentration measured by absorption at 450nm.

Results: CYP1B1 was expressed best in pCWori (+). It does not express in LB-media and requires TB for expression.1:1 mixture of peptone/tryptone based TB showed best expression (with 1% glucose). Highest yields were seen after 24 hours (250nmol/l). Higher IPTG conc enhanced expression but was ineffective beyond 0.6mM. CYP1B1 was best expressed at 30°C. Trace element solution is indispensible. Thiamine did not show enhancement but 5-ALA proved to be useful (best at 0.8mM). Phosphate buffer system enhanced expression.

Conclusions: All functional studies done on modified CYP1B1 are invalid.We report the development of a protocol which reveals that culture in TB media (1:1 peptone + tryptone), PBS (pH7.2), trace elements solution, ALA (0.8mM) at 30°C and 150 RPM yields unmodified CYP1B1 with consistency and repeatability.

Keywords: 539 genetics • 735 trabecular meshwork  
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