Beta-lactamase+A,+Yersinia+enterocolitica

Citation: https://www.cdc.gov/yersinia/
 * Target (protein/gene name)**: Beta-lactamase A (blaA)
 * NCBI Gene # or RefSeq#:** 4715765
 * Protein ID (NP or XP #) or Wolbachia#:** YP_001006271.1
 * Organism (including strain):** from //Yersinia enterocolitica//
 * Etiologic Risk Group:** Category B Priority Pathogens
 * Disease Information:** //Yersinia enterocolitica// is a bacterium that leads to enterocolitis in humans, as well as yersiniosis and sepsis. //Yersinia enterocolitica// operates with the help of two beta-lactamases, A and B, which contribute to this bacterium's resistance to penicillin and cephalosporins. Although these diseases tend to self-treat themselves, they result in unpleasant symptoms like diarrhea and may lead to an increased risk for arthritis due to the immune system's response to this drug. The bacterium is also extremely widespread and leads to over 100,000 known cases per year.
 * Link to TDR Targets page (if present):** N/A
 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.):** https://www.ncbi.nlm.nih.gov/gene/4715765
 * Essentiality of this protein:** Could not find information attesting to the direct link between the survival of the bacteria and this protein, but multiple studies have found that beta-lactamase A is primarily important in bacteria to protect them from being killed by antibiotics. This indicates that beta-lactamases are essential to bacterial survival, albeit indirectly, so inhibiting it will lead to successful elimination of these bacteria.
 * Is it a monomer or multimer as biological unit? (make prediction at @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html):** According to the structure analysis, the protein appears to exist as monomers.
 * Complex of proteins?:** Yes
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):** Beta-lactamase A and other beta-lactamases are druggable targets that have many different inhibitors for the varieties that exist in many different organisms. The issue is that antibiotic resistance develops quickly so new inhibitors are in constant demand. Citation: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806661/


 * EC#**: 3.5.2.6
 * Link to BRENDA EC# page**: http://www.brenda-enzymes.info/enzyme.php?ecno=3.5.2.6
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):** colorimetric assay for detecting enzyme activity with absorbance at 490 nm; user needs to provide spectrophotometer, clear plate with many wells, and dimethyl sulfoxide (DMSO)
 * Link to Sigma (or other company) page for assay** **or link (or citation) to paper that contains assay information:** https://www.ncbi.nlm.nih.gov/pubmed/18437319
 * Links to assay reagents (substrates) page:** http://www.abcam.com/beta-lactamase-activity-assay-kit-colorimetric-ab197008.html
 * Cost and quantity of substrate reagents, supplier, and catalog #:** 100 tests for $685 + $50 shipping from Abcam, catalog number: ab197008

code 1   MSASSPVSIHPEHLPIRVNAAIDQALAEQRLVGAVVIIALHGEIHFCRAAGLAERETARP  60 MSASSPVSIHPEHLPIRVNAAIDQALAEQRLVGAVVIIALHGEIHFCRAAGLAERETARP Sbjct 1    MSASSPVSIHPEHLPIRVNAAIDQALAEQRLVGAVVIIALHGEIHFCRAAGLAERETARP  60
 * Structure (PDB or Homology model)**
 * PDB # or closest PDB entry if using homology model:** 5E2E
 * For Homology Model option:** N/A
 * Pairwise alignment of your BLASTP search in NCBI against the PDB--99% aligned**

Query 61   MTVDTLFRLASISKPIVSTAAMVLVAQGKLELDGDIRRWLPDFQPRLAQGELADITVRQL  120 MTVDTLFRLASISKPIVSTAAMVLVAQGKLELDGDIRRWLPDFQPRLAQGELADITVRQL Sbjct 61   MTVDTLFRLASISKPIVSTAAMVLVAQGKLELDGDIRRWLPDFQPRLAQGELADITVRQL  120

Query 121  LSHTAGLGYRFSEADDDGPYARAGVSDGMDKSEITLHENLRRLATVPLHFAPGTSWRYSL  180 LSHTAGLGYRFSEADDDGPYARAGVSDGMDKSEITLHENLRRL TVPLHFAPGTSWRYSL Sbjct 121  LSHTAGLGYRFSEADDDGPYARAGVSDGMDKSEITLHENLRRLTTVPLHFAPGTSWRYSL  180

Query 181  AIDVIGAVIEQVCGQPLNQAIRALVTGPLQMNDTDFFTRDIGRLAPPYVNDLPAPRLLQE  240 AIDVIGAVIEQVCGQPLNQAIRALVTGPLQMNDTDFFTRDIGRLAPPYVNDLPAPRLLQE Sbjct 181  AIDVIGAVIEQVCGQPLNQAIRALVTGPLQMNDTDFFTRDIGRLAPPYVNDLPAPRLLQE  240

Query 241  GECVAFSPEEIGVRFSPARALNAQSFPSGGAGMVGSASDLLRLLETLRQGGAPLLPAALV  300 GECVAFSPEEIGVRFSPARALNAQSFPSGGAGMVGSASDLLRLLETLRQGGAPLLPAALV Sbjct 241  GECVAFSPEEIGVRFSPARALNAQSFPSGGAGMVGSASDLLRLLETLRQGGAPLLPAALV  300

Query 301  DEMGRDQTGGIPLPDAPGIGFGLGFSVLRNPLEAASPESIGTWSWGGVYGHSWFVDPMQE  360 DEMGRDQTGGIPLPDAPGIGFGLGFSVLRNPLEAASPESIGTWSWGGVYGHSWFVDPMQE Sbjct 301  DEMGRDQTGGIPLPDAPGIGFGLGFSVLRNPLEAASPESIGTWSWGGVYGHSWFVDPMQE  360 Query 361  LSVVALTNTLYEGMSGQFVNDLRDAIYASR  390 LSVVALTNTLYEGMSGQFVNDLRDAIYASR Sbjct 361  LSVVALTNTLYEGMSGQFVNDLRDAIYASR  390 code
 * Query Coverage:** 99%
 * Max % Identities:** 100
 * % Positives:** 389/390
 * Chain used for homology:** serine hydrolase

citation: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806661/ code format="genbank" 1 msasspvsih pehlpirvna aidqalaeqr lvgavviial hgeihfcraa glaeretarp 61 mtvdtlfrla siskpivsta amvlvaqgkl eldgdirrwl pdfqprlaqg eladitvrql 121 lshtaglgyr fseadddgpy aragvsdgmd kseitlhenl rrlatvplhf apgtswrysl 181 aidvigavie qvcgqplnqa iralvtgplq mndtdfftrd igrlappyvn dlpaprllqe 241 gecvafspee igvrfspara lnaqsfpsgg agmvgsasdl lrlletlrqg gapllpaalv 301 demgrdqtgg iplpdapgig fglgfsvlrn pleaaspesi gtwswggvyg hswfvdpmqe 361 lsvvaltntl yegmsgqfvn dlrdaiyasr code ATGAAGCACTCTTCGCTACGGCGTTCACTATTATTAGCCGGTATTACCCTGCCCTTAGTCAATTTTGCGCTGCCA ACCTGGGCCGCTGCGATTCCGGGATCATTAGACAAGCAATTGGCAGCACTTGAGCATAGTGCTAACGGTCGTT TAGGCATTGCGATGATTAATAGCGGCGCTGGCACCAAAATTTTGTATCGCGGAGCTCAACGTTTCCCATTCTGT AGTACTTTTAAGTTTATGCTGGCCGCTGCTGTATTAGATCAAAGCCAGTCTCAGCCAAATTTACTCAATAAGCATA TAAATTACCATGAGAGTGACTTGTTATCTTATGCCCCGATCACGCGTAAAAATCTGGCGTGTGGCATGACCGTCT CCGAGTTATGTGCTGCCACCATTCAATATAGTGATAACACTGCCGCGAATTTATTAATTAAAGAGTTGGGTGGTTT AGCGGCTGTTAATCAGTTTGCTCGCAGTATTGGCGATCAGATGTTCAGATTAGACCGCTGGGAGCCTGATTTAA ATACCGCGCTACCTAATGACCCACGTGATACCACCACTCCTGCGGCTATGGCAGCGAGTATGAATAAATTGGTG CTGGGTGATGCATTGCGCCCTGCCCAACGAAGTCAACTCGCTGCATGGCTGAAAGGAAATACCACCGGGGATG CCACAATTCGTGCGGGTGCCCCTACTGACTGGATTGTGGGTGACAAAACGGGTAGTGGCGATTACGGAACCAC CAATGATATTGCGGTACTTTGGCCGACTAAAGGCGCGCCGATTGTTTTAGTCGTGTATTTCACGCAACGCGAAAA AGACGCGAAGCCACGGCGCGATGTATTGGCCTCTGCGACCCAAATAATTTTGTCGCAAATATCCTGA
 * Current Inhibitors:** various β-lactam antibiotics although these are frequently becoming ineffective because of antibiotic resistance developing:
 * Expression Information (has it been expressed in bacterial cells):** Yes, it has been expressed in recombinant //E. coli// JM103; citation: https://www.ncbi.nlm.nih.gov/pubmed/18588109
 * Purification Method:** Beta-lactamase can be purified using ammonium sulfate and chelating with Zn2+ in a manner similar to the Ni2+ resin that we used in our lab. As for characterization, an SDS-PAGE gel can also be used. citation: https://www.ncbi.nlm.nih.gov/pubmed/11570857
 * Image of protein (PyMol with features delineated and shown separately):**
 * Amino Acid Sequence:**
 * length of your protein in Amino Acids:** 390 aa
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website:** 41.85 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 0.906
 * TMpred graph Image** (@http://www.ch.embnet.org/software/TMPRED_form.html).
 * CDS Gene Sequence:**
 * GC% Content for gene:** 218 G, 210 C, and 885 total bases, so 48.36%
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):** N/A
 * GC% Content for gene (codon optimized)**: N/A

don't need this for Sp18. Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.

Primer design results for 'tail' primers (this is just 2 sequences):