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Determination of Methods to Separate and Analyze Small and Large Chain Isoprenoids from Mycobacterium tuberculosis
Author(s) -
Guthrie Allison,
Mann Francis
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.884.49
Subject(s) - terpenoid , biochemistry , mycobacterium tuberculosis , chemistry , high performance liquid chromatography , gene , biosynthesis , escherichia coli , chromatography , biology , tuberculosis , medicine , pathology
The genes responsible for the isoprenoid carrier lipids involved in cell wall biosynthesis of Mycobacterium tuberculosis (M.tb) are believed to be Rv2361c, Rv1086, and Rv0989c. These genes encode prenyltransferases catalyzing production of isoprenoids of increasing length. Rv0989c and can work in tandem with Rv1086 to create precursors to decaprenyl diphosphate, the final product of Rv2361c. Rv2361c can also work by itself to produce decaprenyl diphosphate. In addition to Rv2361c, Rv1086, and Rv0989c, M.tb encodes seven total different prenyltransferases, and in other biochemical studies it has been suggested that these genes may provide redundancy with the isoprenoids that are formed. These genes can be expressed in Escherichia coli and the isoprenoids extracted through lipid extraction. The samples are then dephosphorylated using alkaline phosphatase. Once dephosphorylated these samples can be analyzed using HPLC in order to separate and analyze the isoprenoid products. HPLC separation occurs with a mobile phase of water and an increasing gradient of 60:40 isopropanol:methanol at a rate of 1 mL/min. These samples are ran over a C18 column. The HPLC allows us to view the different products that may be formed during errant metabolism when alternate isoprenoid precursors are available. Continuing work with these products will allow us to see the influence of each gene with the other genes present in M.tb in the formation of decaprenyl diphosphate.