Endoproteinase Lys

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...

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-... 

Endoproteinase Lys-C (Lysobacter enzymogenety Lys-X pH 8.5 TLCK, aprotinin. leupeptin... 

The resulting solution was diluted with 75 pi of water and endoproteinase Lys-C at 1% by weight was added. The sample was incubated at 37 C for 20 hr before it was quenched with TFA. The peptides generated were separated by chromatography through a Vydac C18 column (0.21 x 5 cm). The solvents and the gradient used were identical to those described above except the flow rate was changed to 0.25 ml per min. 

Figure 4 Endoproteinase Lys-C map of mature NT-3 (A) peptide map of the higher molecular weight form (B). The expected C-terminal fragment I1I6GRT119 of the mature protein is highlighted in bold.

As shown in Table I, 76% of the 39 laboratories that participated in this study routinely carry out in situ PVDF and/or in-gel digests, and trypsin (78%) or endoproteinase Lys-C (43%) are the two most frequently used enzymes. The most commonly cited protocols that were routinely used included those by... 

Enzymatic digestion was performed at a 1/20 (w/w) ratio of each enzyme (Endo-Lys C and chymotrypsin) to substrate. Endoproteinase Lys-C (Endo Lys-C, Wako) digestion was performed according to Riviere et al. (1991). The protein... 

Limited proteolysis bv endoproteinase Lvs-C Sample was reconstituted in 20 mM Tris-HCl buffer, pH 7.5 (1 mg/ml) and digested with endoproteinase Lys-C (enzyme-to-substrate ratio= 1 100) at 25 C. At 15 min and 2 h, sample aliquots (1(K) il each) were taken and digestion stopped by adding 5 j,l of 20% TFA. Samples of 5-20 p,g were dried completely and subjected to SDS-PAGE as described below. 

A. Limited endoproteinase Lys-C digestion Figure 4A shows SDS-PAGE of digests generated by limited proteolysis (nonreducing conditions, for 15 min [lane 1] or 2 h [lane 2]) with Lys-C protease. The SCF polypeptide has... 

Figure 4. SDS-PAGE of peptide products of SDS-nondissociable dimer derived from chemical and proteolytic cleavages. A, endoproteinase Lys-C digestion. Lanes 1 and 2 (nonreducing), products at 40 and 10 pg lanes 3 and 4, as lanes 1 and 2, but reducing. B, CNBr cleavage of Met-oxidized dimer. Lane 1 (nonreducing), oxidized dimer lane 2 (nonreducing), cleavage product lanes 3 and 4, as lanes 1 and 2, but reducing.

Fig. 1. Reversed phase HPLC chromatogram of an endoproteinase Lys C digest of PrP 27-30, showing absorbance at 214 and 280 nm versus time (min). The gradient line shows acetonitrile content. The peptide compositions shown for each peak were derived from the experiments described in Section III. Peptides were identified containing all residues between 74 and 231, with the exception of an anticipated tetrapeptide, residues 107-110. (Reproduced with permission from Stahl, N. et al. in Prusiner, S.B., Collinge, J., Powell, J., and Anderton, B. [1992], Prion Diseases of Humans and Animals, pp. 361-379. Copyright Elsevier Science).

Fig. 3. Chemical and enzymatic treatment to reduce the size and complexity of the GPI anchor for mass spectrometric analysis. PIPLC removed the acylalkylglycerol lipids, then endoproteinase Lys C cut the amino acid chain after Lys220, giving the C-terminal peptide attached to the phosphorylated glycan. Incubation with 50% aqueous HF was used to hydrolyze the phosphodiester bonds and to release the glycan and the peptide, which were separated by RP-HPLC and analyzed independently.

The primary structure was assessed by peptide mapping and N- and C-terminal sequencing. N-terminal sequence analysis showed that a single sequence was detected, MKAIFVLNAA, which corresponds exactly to the first 10 amino acids at the N terminus of P40 as predicted from the DNA sequence. Reverse phase HPLC analysis of a digestion of P40 with a lysine-specific endopeptidase, i.e. endoproteinase Lys-C, was used for identification and primary structure confirmation (Fig. 9). Endoproteinase Lys-C hydolyzes peptide bonds at the carboxylic side of lysine residues. The seventeen peaks resolved were characterized by mass spectrometry, allowing the confirmation of 99 % of the primary sequence. 

When P40 was digested with endoproteinase Lys-C in the presence of the detergent used to refold and purify the protein, several peptides identified by LC-MS analysis contained 3 to 4 theoretical fragments. These peptides were localized in the membrane domain of P40 and resulted from an incomplete digestion of this part of the protein. This was mostly due to the non accessibility of some lysine residues in the P-barrel formed in the presence of the detergent. On the basis of the two-dimensional model of the P-barrel domain of E. coli OmpA (Vogel and Jahnig, 1986 Pautsch and Schulz,... 

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