Endoproteinases

Endoproteinase Lys-C The carboxy terminus side of lysine, except when proline is attached to lysine... 

Table 5.9 Peptides detected during the LC-electrospray-MS (LC-ESMS) analysis of the endoproteinase Lys-C digest from native cytochrome c". Reprinted from Biochim. Biophys. Acta, 1412, Klarskov, K., Leys, D., Backers, K., Costa, H. S., Santos, H., Gnisez, Y. and Van Beenmen, J. J., Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides", 47-55, Copyright (1999), with permission from Elsevier Science...

The digestion of the protein, after heme removal, using Glu-C endoproteinase was also carried out. This enzyme cleaves the polypeptide backbone on the carboxyl terminus of a glutamic acid residue and in this case yielded twelve chromatographic responses. Despite two of these arising from unresolved components, molecular weight information was obtained from 15 polypeptides, one of which was the intact protein, covering the complete sequence, as shown in Table 5.10. 

Such a process led to the full assignment of the sequence of the cytochrome c" protein shown in Figure 5.17, in which the polypeptide fragments generated by enzymatic digestion with endoproteinase Lys-C and Glu-C are also indicated. 

Salutaridinol 7-O-acetyltransferase was purified to apparent electrophoretic homogeneity from P. somniferum cell suspension cultures and the amino acid sequence of ten endoproteinase Lys-C-generated peptides was determined.28 A comparison of these amino acid sequences with those available in the GenBank/EMBL sequence databases indicated no relevant similarity to known proteins. The first attempt to isolate a cDNA encoding salutaridinol 1-0-... 

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

Rush, R. S., Derby, P. L., Strickland, T. W., and Rohde, M. F. (1993). Peptide-mapping and evaluation of glycopeptide microheterogeneity derived from endoproteinase digestion of erythropoietin by affinity high-performance capillary electrophoresis. Anal. Chem. 65, 1834—1842. 

Prabakaran, S., Tepp, W. and DasGupta, B.R., Botulinum neurotoxin types B and E purification, limited proteolysis by endoproteinase Glu-C and pepsin, and comparison of their identified cleaved sites relative to the three-dimensional structure of t q)e A neurotoxin, Toxicon, 39, 1515-1531, 2001. 

Glutamyl endopeptidase [EC 3.4.21.19] (also known as staphylococcal serine proteinase, V8 proteinase, protease V8, and endoproteinase Glu-C), a member of the peptidase family S2B, catalyzes the hydrolysis of Asp-Xaa and Glu-Xaa peptide bonds. In appropriate buffers, the specificity of the bond cleavage is restricted to Glu-Xaa. Peptide bonds involving bulky side chains of hydrophobic aminoacyl residues are hydrolyzed at a lower rate. 

Fig. 5 Screening by affinity CE for interaction of SAP peptides with heparin in solution. An endoproteinase Asp-N-treated Glu-C digest of SAP solubilized in water was injected for 12 s and subjected to CE at 15 kV (detection at 200 nm) in the presence of heparin (Hep) (B) added to the electrophoresis buffer (0.1 M phosphate, pH 7.5) at the concentration indicated. The peptide marked with asterisks was identified by spiking with HPLC-purified fragments and corresponds to the fragment in Figure 6. (From Ref. 71.)...

Melanocyte stimulating hormone (a and (5forms) Endoproteinase with trypsin like activity... 

The oleosin fusion procedure was used for the purification of the commercially valuable plant-based blood anticoagulant hirudin in transgenic Brassica carinata and Brassica napus. Hirudin, a natural protein from the medicinal leech Hirudo medicinalis, is superior to other anticoagulants such as heparin. Recombinant hirudin was cleaved from oil-bodies using endoproteinase Factor Xa. Released hirudin was biologically active, as determined by a colorimetric thrombin inhibition assay. 

Endoproteinase LysC cleaved AP-A between Lys37 and Ala38 to yield a derivative with cardiotonic activity an order of magnitude lower than that of the parent molecule (Monks SA and Norton RS, unpublished results). This reduction in activity could be a consequence of local conformational perturbations. Treatment of AP-B with carboxypeptidase removed Lys49, resulting in only a two-fold reduction in cardiotonic activity (Monks SA and Norton RS, unpublished results). 

TPCK-treated trypsin, TLCK-treated chymotrypsin, and endoproteinase-Glu-C (Staphlococcal V8 protease) are all obtainable from Sigma (Poole, UK)... 

Jones, B.L., Fontanini, D., Jarvinen, M., and Pek-karinen, A. 1998. Simplified endoproteinase assays using gelatin or azogelatin. Anal. Biochem. 263 214-220. 

Resing, K. A., Thulin, C., Whiting, K., al-Alawi, N., and Mostad, S. (1995). Characterization of profilaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase of epidermis./. Biol. Chem. 270, 28193-28198. 

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