Target-New+Delhi+Metallo+Beta+Lactamase

NCBI Gene#: 14858116 NC_021180.1 Klebsiella pneumoniae //For a century, modern society has relied on beta-lactam drugs such as penicillin-G to treat serious gram positive as well as gram negative bacterial infections. However, heavy reliance of these drugs has also selected for beta-lactamase synthesis as a form of an antimicrobial resistance mechanism in bacteria.  Beta-lactamase cleaves the four-membered beta-lactam ring through hydrolysis, rendering it ineffective against infections. In conditions where antibiotic overuse is widespread, this spread of this mechanism via horizontal gene transfer has resulted in deadly versions of minor infections. A recently discovered beta-lactamase, New Delhi metallo beta-lactamase (NDM-1), resists most lactam-based inhibitors and is effective against a wide range of antibiotics of size and shape. //
 * Target (protein/gene name): New Delhi Metallo Beta-Lactamase**
 * NCBI Gene # or RefSeq#:**
 * Protein ID (NP or XP #) or Wolbachia#:**
 * Organism:**
 * Etiologic Risk Group : RG1**
 * Background/Disease Information:**


 * Essentiality of this protein: Essential in the presence of beta-lactam antibiotics such as ampicillin and penicillin to confer resistance**
 * Complex of proteins: There are identical proteins Chain A and B for 3Q6X.**
 * Druggable Target: Druggability index:** 0.5 for Class A Beta Lactamase

Does not exist for NDM-1/K. pneumoniae Following picture has correct reagent and product
 * *EC#: **
 * 3.5.2.6 ||
 * Link to BRENDA EC# page:**
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrometric or Titrimetric assay**
 * Reagents:**

Thomas PW , Zheng M , Wu S , Guo H , Liu D , Xu D , <span style="background-color: #ffffff; color: #660066; font-family: arial,helvetica,clean,sans-serif; font-size: 12px;">Fast W <span style="background-color: #ffffff; font-family: arial,helvetica,clean,sans-serif; font-size: 12px;">. 2011: <span style="background-color: #ffffff; font-family: arial,helvetica,clean,sans-serif; font-size: 12px;">Assay solution: 0.1M Hepes, pH 7, 20uM ZnSO4 and 0.1mM substrate in water. NDM-1 diluted in 50mM Tris-HCl, pH 7, and 150mM NaCl. Substrate: Nitrocefin, Calbiochem, 5mg: $109.00

100 mM Tris HCl Buffer with 0.1% (w/v) Bovine Serum Albumin, pH 7.0 at 25°C Ampicillin/beta-lactam Solution (Control) 10 mM Sodium Hydroxide Solution Standardized (NaOH) **-- link to Sigma (or other company) page for assay or assay reagents** http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/penicillbenzyl.pdf http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024621 157.50 $136
 * -- link (or citation) to paper that contains assay information:**
 * -- List cost and quantity of substrate reagents and supplier**
 * Supplier Sigma Aldrich**
 * Ampicillin**
 * Santa Cruz Biotechnology, inc**
 * Bovine Serum Albumin**-100g

-- PDB # :3Q6X
 * Structure Available (PDB or Homology model)**




 * Current Inhibitors: Aztreonam and possibly Clavulanic Acid**
 * Expression Information (has it been expressed in bacterial cells):**
 * Purification Method: His-6, Nickel or Strep tag**


 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): From Fast document***
 * MELPNIMHPVAKLSTALAAALMLSGCMPGEIRPTIGQQMETGDQRFGDLVFRQLAPNVWQHTSYLDMPGF **
 * GAVASNGLIVRDGGRVLVVDTAWTDDQTAQILNWIKQEINLPVALAVVTHAHQDKMGGMDALHAAGIATY **
 * ANALSNQLAPQEGMVAAQHSLTFAANGWVEPATAPNFGPLKVFYPGPGHTSDNITVGIDGTDIAFGGCLI **
 * KDSKAKSLGNLGDADTEHYAASARAFGAAFPKASMIVMSHSAPDSRAAITHTARMADKLR **

e280 = 28,500 M−1 cm−1 ([|from Kim][| paper])
 * length of your protein in Amino Acids: 270 Amino Acids**
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: 28.6107 kDa**
 * Molar Extinction coefficient of your protein at 280 nm wavelength:**


 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * *CDS Gene Sequence (paste as text only): **


 * atggaattgcccaatattatgcacccggtcgcgaagctgagcaccgcatta 61 **


 * 62 gccgctgcattgatgctgagcgggtgcatgcccggtgaaatccgcccgacgattggccag 121 **


 * 122 caaatggaaactggcgaccaacggtttggcgatctggttttccgccagctcgcaccgaat 181 **


 * 182 gtctggcagcacacttcctatctcgacatgccgggtttcggggcagtcgcttccaacggt 241 **


 * 242 ttgatcgtcagggatggcggccgcgtgctggtggtcgataccgcctggaccgatgaccag 301 **


 * 302 accgcccagatcctcaactggatcaagcaggagatcaacctgccggtcgcgctggcggtg 361 **


 * 362 gtgactcacgcgcatcaggacaagatgggcggtatggacgcgctgcatgcggcggggatt 421 **


 * 422 gcgacttatgccaatgcgttgtcgaaccagcttgccccgcaagaggggatggttgcggcg 481 **


 * 482 caacacagcctgactttcgccgccaatggctgggtcgaaccagcaaccgcgcccaacttt 541 **


 * 542 ggcccgctcaaggtattttaccccggccccggccacaccagtgacaatatcaccgttggg 601 **


 * 602 atcgacggcaccgacatcgcttttggtggctgcctgatcaaggacagcaaggccaagtcg 661 **


 * 662 ctcggcaatctcggtgatgccgacactgagcactacgccgcgtcagcgcgcgcgtttggt 721 **


 * 722 gcggcgttccccaaggccagcatgatcgtgatgagccattccgcccccgatagccgcgcc 781 **


 * 782 gcaatcactcatacggcccgcatggccgacaagctgcgctga **

60.820045558087%
 * GC% Content for gene:**
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):**


 * GC% Content for gene (codon optimized):**


 * Buffers from** Shen 2013

Harvested cells by centrifugation at 10000xg for 15 min at 4 C, suspended in 30 mM phosphate-buffered saline, pH 7.3

Lysis: 30 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.3 Wash: 30 mM Tris-HCl, 0.5 M NaCl, 40 mM imidazole, pH 7.3 Elution: 30 mM Tris-HCl, 0.5 M NaCl, 100 mM imidazole, pH 7.3

Did Ni-NTA twice, then dialyzed against 2 L of 20 mM HEPES, 100 uM Zn(NO3)2, pH 6.8 overnight at 4 C.