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Protein – H pylori IMPDH

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Protein – H pylori IMPDH

HpIMPDH, Active(Recombinant enzyme expressed in E. coli Rosetta-gami™ 2 (DE3) pLysS competent cells)

Product Description: N-terminal 6Histagged, recombinant, H. pylori inosine-5’-monophosphate dehydrogenase (HpIMPDH) full length, expressed in E. coli Rosetta-gami™ 2 (DE3)pLysS competent cells. Purified using immobilized metal affinity chromatography. Purified by SDS-PAGE and Coomassie blue staining. Molecular Weight = 55.5 kDa.
Specific Activity: Approximately 120 U/mg, specific activity is defined as the nmol of NADH produced per minute per mg of HpIMPDH at 37°C with a final IMP concentration of 250 µM and NAD+ concentration of 300 µM.

Formulation: 1 mg/ml of enzyme in 40 mM Tris – HCl pH 7.8, 150 mM NaCl and 2 mM DTT. Frozen solution.
Storage and Stability: On receipt of material store at -70°C. Unopened enzyme is stable for up to 1 month from date of shipment when stored at recommended storage temperature. Avoid repeat freeze/thaw cycles. For maximum recovery of product, centrifuge original vial prior to removing the cap.
Handling Recommendations: Rapidly thaw the vial under cold water and immediately place on ice. Aliquot unused material into pre-chilled micro-centrifuge tubes and immediately snap-freeze the vials in liquid nitrogen prior to re-storage at -70°C.

Quality Control Testing

HpIMPDH Assay: 1.11 µg of HpIMPDH reduces NAD+ to NADH in the assay protocol described in page 2. The specific activity is determined by subtracting the assay controls.

MS Tryptic Fingerprint: Confirmed identity as HpIMPDH with the translated sequence listed on pages 2 and 3.

SDS-PAGE and Coomassie Stain

Purity of the enzyme was assessed by SDS-PAGE and Coomassie blue staining.

HpIMPDH assay protocol

1. 1X Reaction buffer: 50 mM Tris-Cl pH 7.8, 100 mM KCl and 3 mM DTT
2. 2X Substrate buffer: 500 µM IMP and 600 µM NAD+ in 1X reaction buffer
3. 2X Assay buffer: 200 nM HpIMPDH in 1X reaction buffer
4. Mix 100 µL each of substrate buffer and assay buffer in a 96-well black plate to initiate the reaction at 37 °C.
5. Dilute 2X substrate buffer and 2X assay buffers to their 1X concentrations separately in the 1X reaction buffer, and use as controls for the reaction.
6. Measure the specific activity of the enzyme in terms of the fluorescence of NADH (λem = 440 nm and λex = 340 nm).
7. Subtract the reaction reading from the controls to calculate the specific activity of the enzyme.

HpIMPDH Sequence Information

Protein – H. pylori inosine-5’-monophosphate dehydrogenase (HpIMPDH; H. pylori IMP dehydrogenase)
Tag – N-terminal 6X histidine
Accession number – WP_001223726.1

HpIMPDH amino acid sequence (UniProt ID: P56088):

1      MRILQRALTFEDVLMVPRKSSVLPKDVSLKSRLTKNIRLNIPFISAAMDTVTEHKTAIAM
61    ARLGGIGIVHKNMDIQTQVKEITKVKKSESGVINDPIFIHAHRTLADAKVITDNYKISGV
121  PVVDDKGLLIGILTNRDVRFETDLSKKVGDVMTKMPLVTAHVGISLDEASDLMHKHKIEK
181  LPIVDKDNVLKGLITIKDIQKRIEYPEANKDDFGRLRVGAAIGVGQLDRAEMLVKAGVDA
241  LVLDSAHGHSANILHTLEEIKKSLVVDVIVGNVVTKEATSDLISAGADAIKVGIGPGSIC
301  TTRIVAGVGMPQVSAIDNCVEVASKFDIPVIADGGIRYSGDVAKALALGASSVMIGSLLA
361  GTEESPGDFMIYQGRQYKSYRGMGSIGAMTKGSSDRYFQEGVASEKLVPEGIEGRVPYRG
421  KVSDMIFQLVGGVRSSMGYQGAKNILELYQNAEFVEITSAGLKESHVHGVDITKEAPNYYG

HpIMPDH nucleotide sequence (UniProt ID: P56088):

atgagaattt tacaaagggc tttgactttt gaagatgtgt tgatggtgcc tagaaagtct         60
agcgttttac ctaaagatgt gagcttaaag tctcgcttaa ctaaaaacat tcgtttgaat         120
atccccttta tcagtgcggc tatggatacg gttacagagc ataaaaccgc tatcgctatg      180
gcgcgccttg ggggtattgg catcgtgcat aaaaacatgg atattcaaac gcaagttaaa    240
gaaatcacta aggttaaaaa aagcgagagc ggggtgatta atgatcctat ttttatccat       300
gcgcacagga cgctagcgga cgctaaagtc ataacggata attacaagat ttcaggcgtg  360
cctgtggtag atgataaggg gttgttgatt gggattttaa ccaacagaga tgtgcgcttt         420
gaaaccgatt tgagtaaaaa agtgggcgat gtgatgacta aaatgccttt agttaccgct      480
catgtgggta tcagtttgga tgaagcgagc gatttgatgc acaagcataa gattgaaaaa     540
ttgcccattg tggataaaga taatgtctta aaaggcttga tcacgatcaa agatattcaa        600
aaacgcattg aataccctga ggccaataaa gatgattttg ggaggttgag agtgggggcg   660
gctattggag tggggcagtt ggatagggct gagatgttag ttaaagcggg ggtggatgca    720
ctggtgctag acagcgcaca tgggcattca gccaatatct tacacacttt agaagagatt     780
aaaaaaagct tggtagtgga tgtgattgtg gggaatgtgg ttactaaaga agccacaagc   840
gatttgatta gcgcgggagc agacgctatt aaagtgggta ttgggccagg aagcatttgc    900
accactagga ttgtggctgg ggtgggaatg ccccaagtga gcgcgattga taattgcgta   960
gaagtggcgt ctaaatttga tattcctgtg attgcagatg gagggatccg ctattcaggc      1020
gatgtggcta aggctttggc tttgggggca tcaagcgtga tgataggctc tttactcgct      1080
ggcacagaag aatctcctgg ggattttatg atctatcaag ggaggcaata taaaagctat   1140
aggggcatgg gcagcattgg ggctatgact aaagggagct ctgataggta ttttcaagag   1200
ggcgtagcga gtgaaaagtt agtcccagaa ggcattgaag ggcgtgtgcc ttatcgtggt   1260
aaggtttcgg atatgatttt ccaattagta gggggcgtgc gctcttctat ggggtatcag         1320
ggggcgaaaa atattttgga attgtatcaa aacgctgaat ttgtagaaat cactagcgcg      1380
gggttaaaag aaagccatgt gcatggcgtg gatattacta aagaagcccc taattattat      1440
gggtga                                                                                                                    1446

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