TargetSp17+-+Ricin+Toxin+Chain+A+&+B+(Ricinus+communis)

Ricin Toxin Chain A/B:
Ricin is a protein produced by the plant Ricinius communis (Castor bean) and is one of the deadliest chemical compounds in the world with LD50 as low as 1-2ug/kg if injected, 21-43ug/kg if inhaled, and 1-20mg/kg if ingested. The plant is native to Africa but found all over the world due to its industrial, cosmetic, and ornamental uses. It produces Ricin in it's leaves and particularly in its seeds which are used to make castor oil a particular common oil (Ricin is water soluble and does not make it to the oil). Due to it's availability it has been used in multiple murders and is a likely biological weapon. It's symptoms begin to show at different times depending on the method of uptake anywhere from 3-36 hours. Symptoms include: bleeding, diarrhea, vomiting, abdominal pain, low blood pressure, and dehydration. It is a 2 chain protein with B allowing access to the cell and A causing symptoms. Chain A inhibits ribosomes by changing the nucleotide base in the ribosome whether in the ribosome or by changing the rRNA in a highly conserved sequence. The simple nature of the change allows it to inhibit 1500 ribosomes a minute. [[|1]] Chain B binds to surface carbohydrate receptors while maintaining normal function of the receptors as well. Applications of better understanding Ricin are not limited to bioweapon prevention but potential targeting of cancer cell ribosomes.
 * *Target (protein/gene name): **Ricin
 * *NCBI Gene # : **LOC8261245
 * *Protein ID: **2AAI
 * *Organism (including strain): **Ricinus communis (castor bean)
 * Etiologic Risk Group (see link below): **[|BSL1]
 * */ Disease Information (sort of like the Intro to your Mini Research Write up): **

http://robertus.cm.utexas.edu/papers/rta-pta.pdf http://www.rcsb.org/pdb/explore/literature.do?structureId=2AAI https://pdb101.rcsb.org/motm/161
 * Link to TDR Targets page (if present): **N/A
 * Link to Gene Database: **https://www.ncbi.nlm.nih.gov/gene/8261245
 * Essentiality of this protein: **Both chains are essential to function.
 * Complex of proteins?: **Yes, chain A and B
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **

[] - assayable in multiple ways Vector labs Chain A/B - $165/mg A&B $150/mg
 * *EC#: **3.2.2.22
 * Link to BRENDA EC# page: **http://www.brenda-enzymes.org/enzyme.php?ecno=3.2.2.22
 * -- ** Show screenshot of BRENDA enzyme mechanism schematic (?)
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **

-- PDB #: 2AAI
 * Structure (PDB or Homology model) **


 * Current Inhibitors: **No current treatment, developing inhibitors.
 * Expression Information (has it been expressed in bacterial cells): ** [|High levels can be produced and simply purified. As well as readily be recombined with Chain B to produce whole ricin]

The clear lysate was precipitated with ammonium sulfate (40% saturation) and the pellet was dissolved and dialysed against Buffer A (10 mM Tris, pH 7.4, 100 mM KCl). The dialysate was filtered through a 0.45-pm filter and loaded onto a strong anion-exchange column Q Sepharose Fast Flow (2.6 X 10 cm), which had been preequilibrated with Buffer A. Unbound proteins were washed off the column with Buffer A and the bound fraction was eluted with Buffer B (Buffer A containing 1.0 M NaCl). Proteins eluted from the column as well as the unbound fraction were analyzed on a 12% SDSPAGE. code >2AAI:A|PDBID|CHAIN|SEQUENCE IFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQRFILVELSNHAELSVTLALDVTNAY VVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQ LPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTAIQESNQGAFASPIQLQRRNGSKF SVYDVSILIPIIALMVYRCAPPPSSQF >2AAI:B|PDBID|CHAIN|SEQUENCE ADVCMDPEPIVRIVGRNGLCVDVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTTYGYSPGVYVMIYDC NTAATDATRWQIWDNGTIINPRSSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGLCLQANSGQVW IEDCSSEKAEQQWALYADGSIRPQQNRDNCLTSDSNIRETVVKILSCGPASSGQRWMFKNDGTILNLYSGLVLDVRASDP SLKQIILYPLHGDPNQIWLPLF code code format="genbank" 1 tcatttcttt aaacttctta catttttttt tgtagcattc tttgtaagtg gaatgacaaa 61 accgttaatg atgttctttt aaaagtgaaa gatgtttata tattgcagta cagataatga 121 tatatctact gcactacata aaacaattta aatctccctg tttattttaa gaagttatat 181 tttctttctt tctcatccta agaaagttaa attactgtaa tcgacattat atgaatttta 241 actaattccg tttctaattt ataattattt cgttaaacca atcaattccc tttaaacact 301 gcttatgcat attctgtctc aatttatata tggcatgcat cttccgtatt aatttataag 361 ttcattttta ttgatcaagt atttgtggtt ttctttatat aaaaaaatgt attagtgttt 421 ttctgtatta attttataag ttcatcttta tgagaatgct aatgtatttg gacagccaat 481 aaaattccaa gaattgctgc aatcaaagat gaaaccggga ggaaatacta ttgtaatatg 541 gatgtatgca gtggcaacat ggctttgttt tggatccacc tcagggtggt ctttcacatt 601 agaggataac aacatattcc ccaaacaata cccaattata aactttacca cagcgggtgc 661 cactgtgcaa agctacacaa actttatcag agctgttcgc ggtcgtttaa caactggagc 721 tgatgtgaga catgaaatac cagtgttgcc aaacagagtt ggtttgccta taaaccaacg 781 gtttatttta gttgaactct caaatcatgc agagctttct gttacattag ccctggatgt 841 caccaatgca tatgtggtcg gctaccgtgc tggaaatagc gcatatttct ttcatcctga 901 <span class="ff_line">caatcaggaa gatgcagaag caatcactca tcttttcact gatgttcaaa atcgatatac 961 <span class="ff_line">attcgccttt ggtggtaatt atgatagact tgaacaactt gctggtaatc tgagagaaaa 1021 <span class="ff_line">tatcgagttg ggaaatggtc cactagagga ggctatctca gcgctttatt attacagtac 1081 <span class="ff_line">tggtggcact cagcttccaa ctctggctcg ttcctttata atttgcatcc aaatgatttc 1141 <span class="ff_line">agaagcagca agattccaat atattgaggg agaaatgcgc acgagaatta ggtacaaccg 1201 <span class="ff_line">gagatctgca ccagatccta gcgtaattac acttgagaat agttggggga gactttccac 1261 <span class="ff_line">tgcaattcaa gagtctaacc aaggagcctt tgctagtcca attcaactgc aaagacgtaa 1321 <span class="ff_line">tggttccaaa ttcagtgtgt acgatgtgag tatattaatc cctatcatag ctctcatggt 1381 <span class="ff_line">gtatagatgc gcacctccac catcgtcaca gttttctttg cttataaggc cagtggtgcc 1441 <span class="ff_line">aaattttaat gctgatgttt gtatggatcc tgagcccata gtgcgtatcg taggtcgaaa 1501 <span class="ff_line">tggtctatgt gttgatgtta gggatggaag attccacaac ggaaacgcaa tacagttgtg 1561 <span class="ff_line">gccatgcaag tctaatacag atgcaaatca gctctggact ttgaaaagag acaatactat 1621 <span class="ff_line">tcgatctaat ggaaagtgtt taactactta cgggtacagt ccgggagtct atgtgatgat 1681 <span class="ff_line">ctatgattgc aatactgctg caactgatgc cacccgctgg caaatatggg ataatggaac 1741 <span class="ff_line">catcataaat cccagatcta gtctagttt code
 * Purification Method : **
 * Image of protein (PyMol with features delineated and shown separately): **
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **
 * *length of your protein in Amino Acids **267
 * Molecular Weight: ** 58859.51
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 89895
 * 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): **
 * *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): **

-- 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 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)

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