Lactate+dehydogenase+(Cryptosporidium+parvum)


 * *Target (protein/gene name): ** Lactate dehydrogenase


 * *NCBI Gene # or RefSeq#: **[|2FN7_A]


 * *Protein ID (NP or XP #) or Wolbachia#: **4ND4


 * *Organism (including strain): **Cryptosporidium parvum (strain Iowa II)


 * Etiologic Risk Group (see link below): **RG2

Cryptosproridiosis is a parasitic disease caused by Cryptosporidium, a genus of protozoan parasites, that is considered to be the most important waterborne pathogen in developed countries. It primarily affects the distal small intestine and the respiratory tract, resulting in watery diarrhea and coughing respectively. The primary symptoms are diarrhea, anorexia, nausea/vomiting, and abdominal pain. The disease can be present in both immunocompetent and immunocompromised individuals with a particular fatal risk for immunocompromised individuals. It is spread through the fecal-oral route most often through ingestion of contaminated water or through coming into contact with contaminated feces. Recent evidence suggests that it is also transmitted via fomites in respiratory secretions. Infection involves ingesting resilient microbial cysts that release sporozoites which then infect the intestinal epithelial tissue. Infection involves the sporozoites attaching to the microvilli of the epithelial cells of the small intestine and then reproduce asexually in a process known as schizogony. These progeny can either develop into Type I merozoites or Type II meronts. Type I continues the infection cycle by attaching onto epithelial cells. Type II meronts contain 4 merozoites which can either be male or female that can reproduce sexually into two types of zygotes. 20% develop into oocysts with thin walls to allow for reinfection while the rest develop into oocysts that are secreted into the environment. Currently, nitazoxanide is the only antiparasitic drug treatment with proven efficacy. There is also research involving the targeting of a oocyst surface protein that has produced a positive response in a large group of cows which is promising for possible human vaccination.
 * */ Disease Information : **


 * Link to TDR Targets page (if present): ** None


 * Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): [|NCBI] **


 * Essentiality of this protein: ** //C.// parvum primarily relies on glycolysis for energy production. Lactate dehydrogenase is essential as a major regulator of glycolysis because the inhibition of the enzyme will cause the loss of energy production.

Is it a monomer or multimer as biological unit ** ? (make prediction at ** @http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer as a biological unit.


 * Complex of proteins?: ** No, since it is a monomer.

Gossypol by competitive inhibition with NADH. [|https://www.sciencedirect.com/science/article/pii/S014181301400823X?via%3Dihub#bib0045]
 * Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): **


 * *EC#: ** 1.1.1.27

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 * Link to BRENDA EC# page: **


 * -- ** Show screenshot of BRENDA enzyme mechanism schematic
 * Figure 1** - BRENDA reaction mechanism of lactate dehydrogenase (E.C. 1.1.1.27).

[] [] [] Supplier: Sigma Aldrich LDH Assay Buffer 50 mL Catalog Number MAK066A LDH Substrate Mix 1 vial Catalog Number MAK066B NADH Standard, 0.5 umole 1 vial Catalog Number MAK066C LDH Positive Control 1 vial Catalog Number MAK066D Individual reagents' prices not listed. -- PDB # or closest PDB entry if using homology model: 4ND4
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): **
 * -- link to Sigma (or other company ) page for assay (see Sigma links below) **
 * -- -or link (or citation) to paper that contains assay information **
 * -- links to assay reagents (substrates) pages. **
 * --- List cost and quantity of substrate reagents, supplier, and catalog # **
 * Structure (PDB or Homology model) **
 * Current Inhibitors: **None
 * Expression Information (has it been expressed in bacterial cells): **CpLDH gene was amplified with PCR and cloned into the pET21a vector. Transformed E. coli strains Rosetta*DE3)pLysS and BL21(DE3) were screened against using ampicillin.
 * Purification Method : ** Size exclusion chromatography

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MIERRKIAVIGSGQIGGNIAYIVGKDNLADVVLFDIAEGIPQGKALDITHSMVMFGSTSKVI
 * Image of protein (PyMol with features delineated and shown separately): **
 * Figure 2 ** - PyMol representation of 4ND4 Chain A shown as sticks colored by element with carbon green and shown as surface with transparency set to 80%.
 * *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): **

GTNDYADISGSDVVIITASIPGRPKDDRSELLFGNARILDSVAEGVKKYCPNAFVICITNPL

DVMVSHFQKVSGLPHNKVCGMAGVLDSSRFRTFIAQHFGVNASDVSANVIGGHGDGM

VPATSSVSVGGVPLSSFIKQGLITQEQIDEIVCHTRIAWKEVADNLKTGTAYFAPAAAAVK

MAEAYLKDKKAVVPCSAFCSNHYGVKGIYMGVPTIIGKNGVEDILELDLTPLEQKLLGESI

NEVNTISKVLDNAPAAGA 1 mierrkiavi gsgqiggnia yivgkdnlad vvlfdiaegi pqgkaldith smvmfgstsk 61 vigtndyadi sgsdvviita sipgrpkddr sellfgnari ldsvaegvkk ycpnafvici 121 tnpldvmvsh fqkvsglphn kvcgmagvld ssrfrtfiaq hfgvnasdvs anvigghgdg 181 mvpatssvsv ggvplssfik qglitqeqid eivchtriaw kevadnlktg tayfapaaaa 241 vkmaeaylkd kkavvpcsaf csnhygvkgi ymgvptiigk ngvedileld ltpleqkllg 301 esinevntis kvldnapaag a
 * *length of your protein in Amino Acids: ** 321 aa
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam] website: ** 33849.94 g/mol
 * Molar Extinction coefficient of your protein at 280 nm wavelength: ** 15930
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
 * Figure 3 ** - TMpred output for amino acid sequence of protein 4ND4 chain A showing its prediction of membrane-spanning regions and their orientation.
 * *CDS Gene Sequence (paste as text only): **


 * *GC% Content for gene: ** 39.447732%
 * *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): **
 * *GC% Content for gene (codon optimized): **

Do Not Need this info for Spring (but still copy these lines to your Target page for now) -- 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): **