Protein Name: Glyoxysomal Malate Dehydrogenase (WMg MDH)
Organism: Watermelon (Citrullus lantus) Plasmid Name: pQE60 wgMDH
Clone/Plasmid History: Originally cloned by into pQE60 vector with the His affinity tag on the C terminus of MDH. This is the mature watermelon glyoxysomal MDH (WMgMDH) without presequence. This construct does NOT have the transit vector and instead has an added Met as the start codon after removal of transit peptide sequence. WMgMDH, was prepared by PCR using the Ncol- and Bglll-site and cloned into the same vector. WMgMDH is cloned between the restriction sites Ncol and BgllI; the Ncol-site also provided the start codon. The necessary restriction sites at the 5'-end and 3'- end of the cDNA sequence were added by PCR. Either during cloning or in subsequent manipulations, the NcoI site (C/CATGG) was mutated to CTATGG, destroying the NcoI cut site.
NCBI / Gene Accession Number: https://www.ncbi.nlm.nih.gov/nuccore/M33148 (shown with transit peptide)
Plasmid Map: Downloadable SnapGene file will include:
· Resistance, Promotor (for bacterial or mammalian), Sequencing primers, RBS and Kozak sequence, History of cloning, Annotated start and stop of protein, Highlighted tags or TEV/Thrombin sites
NCBI Protein Sequence Accession:
Precursor form: https://www.ncbi.nlm.nih.gov/protein/1SEV_A (shown with precursor transit peptide)
Mature form: https://www.ncbi.nlm.nih.gov/protein/1SMK_A (Mature form lacks transit peptide
UniProt Protein Page: https://www.uniprot.org/uniprotkb/P19446/entry
Clone/Protein FAQ and Important Points: High protein expression at 37oF 1mm IPTG for 3-4 hour induction. ~0.25-0.8 mg per ml of culture. pQE60 (Qiagen) is a low copy plasmid and will not give high yields of DNA preps. Amp Resistant. Do not freeze thaw purified protein – stability test of proteins in glycerol needed. Stable at 4oC for several days in elution buffer with minimal loss of activity. Stable at 4oC for 1-4 weeks dialyzed against (10 mM KPi,150 mM NaCl, 0.1 mM EDTA, pH 8.0). Long term storage
-20 to -80oC (10 mM K-phosphate, 0.1 mM EDTA, 20% glycerol, pH 8.0). Concentrations approaching 1-1.25 mg/ml will precipitate over a short time. Dilute immediately after purification and before dyalysis to 1 mg/ml or less. See MDH Stability Datasheet for more information. Expected Km 146µM for NADH and 76 µM for OAA, (additional info from key publications – see above). Purification easily performed in column or batch format.
Crystal Structures:1SEV.pdb, 1SMK.pdb. RCSB PDB Page: https://www.rcsb.org/structure/1SEV ( transit peptide not seen in crystal structure) https://www.rcsb.org/structure/1SMK (mature form)
Key amino acids / functions studied include (shown in the context of other Isoforms):
A landmarks .pse file can be downloaded for use in PyMol showing these regions
Key Publications:
Gietl C, Seidel C, Svendsen I. Plant glyoxysomal but not mitochondrial malate dehydrogenase can fold without chaperone assistance. Biochim Biophys Acta. 1996 May 20;1274(1-2):48-58. doi: 10.1016/0005-2728(96)00009-6. PMID: 8645694.
Cox B, Chit MM, Weaver T, Gietl C, Bailey J, Bell E, Banaszak L. Organelle and translocatable forms of glyoxysomal malate dehydrogenase. The effect of the N-terminal presequence. FEBS J. 2005 Feb;272(3):643-54. doi: 10.1111/j.1742-4658.2004.04475.x. PMID: 15670147.
Available Mutations: Over 80 prepared. See MCC website for list.