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Protease-resistant streptavidin

A technology of streptavidin and trypsin, applied in the field of streptavidin molecules, can solve problems such as large full-length proteins

Inactive Publication Date: 2018-07-17
EURO LAB FUER MOLEKULARBIOLOGIE EMBL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, full-length proteins are often too large for mass spectrometry analysis

Method used

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  • Protease-resistant streptavidin
  • Protease-resistant streptavidin
  • Protease-resistant streptavidin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] Example 1: Reductive methylation of lysine residues in streptavidin

[0242] The chemistry used to block lysine residues is based on the reductive methylation method described by Boersema et al., the modification described in Boersema P.J. et al. (2009) Nat.Protoc.4(4):484-94.

[0243] This blocking reaction is performed with streptavidin already attached to the beads. Subsequently, the biotinylated protein is bound to blocked streptavidin, and the sample is digested with LysC protease, which cleaves the bound protein at lysine residues without exposure to streptavidin.

[0244] Reagent

[0245] 4.5ml of 50mM pH 7.5 Sodium Phosphate Buffer

[0246] 600mM NaBH 3 CN 250μl

[0247] · 4% formaldehyde 250μl

[0248] Acetic acid (Merck, catalog number 1.00063)

[0249] Acetonitrile (ACN) (Biosolve, catalog number 75-05-8)

[0250] · Ammonia solution (25% (vol / vol), Merck, Cat. No. 1.05432)

[0251] · Formaldehyde (CH 2 O) (37% (v / v), Sigma, Cat. No. 252549)

[0252]...

Embodiment 2

[0272] Example 2: Identification of peptides from streptavidin before and after chemical blocking of arginine and lysine

[0273]The lysine and arginine residues in streptavidin are blocked by subsequent reductive methylation and reaction with cyclohexanedione, respectively. The blocking reaction was performed on streptavidin already attached to the beads. Subsequently, the streptavidin beads were trypsinized. The obtained peptides were analyzed by LC-MS.

[0274] Table 1: Tryptic peptides produced by unmodified streptavidin

[0275]

[0276] Tryptic digestion of unmodified streptavidin beads followed by LC-MS analysis identified multiple peptides (see also figure 1 A). A total of 411 profiles were recorded, most of them multiples, indicating their high abundance (Table 1).

[0277] Table 2: Tryptic peptides produced by streptavidin after chemical blocking of K's and R's

[0278]

[0279] After blocking lysine and arginine (by reductive methylation and reaction wit...

Embodiment 3

[0280] Example 3: Identification of peptides from streptavidin before and after chemical blocking of lysine and enzymatic conversion of arginine to citrulline

[0281] The enzyme peptidyl arginine deiminase (Peptidyl arginine deiminase, PAD) can convert arginine to citrulline ( Figure 4 ), while citrulline is not a substrate for trypsin, conferring resistance to proteolytic cleavage.

[0282] This was confirmed by chemically blocking lysine in streptavidin (as in Example 2), followed by enzymatic treatment with PAD to convert arginine to citrulline. This is in stark contrast to the 411 profiles observed after digestion of unmodified trypsin (Table 1), which resulted in the identification of 2 peptides out of a total of 7 profiles (Table 3), and indicated that Chemically modified streptavidin exhibited similar levels of protease resistance (Table 2). Notably, after PAD treatment (Table 3), the arginine-flanked peptide R.NAHSATTWSGQYVGGAEAR.I (SEQ ID NO : 3) The number of sp...

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Abstract

The present invention relates to modified streptavidin molecules that are resistant to cleavage by Lys-C or other proteases. These modified streptavidin molecules can be produced by chemical modification of natural streptavidin, by chemical synthesis or by genetic engineering. The invention also relates to nucleic acid molecules encoding these modified streptavidin molecules, to vectors comprisingsuch nucleic acid molecules, and to cells comprising such nucleic acid molecules or vectors. The invention further relates to solid supports and kits comprising the modified streptavidin molecules. The invention also relates to the use of such modified streptavidin molecules or such solid supports for the capture / immobilization of proteins, peptides, oligonucleotides (e.g. aptamers), polynucleotides (e.g. DNA, RNA, or PNA), lipids, (poly) saccharides, carbohydrates, metabolites, drugs and small molecules, natural and synthetic molecules and to the use of these modified streptavidin moleculesor these solid supports in mass spectrometry for the identification of proteins that interact with aforementioned (bio)molecules. The invention further relates to a method for reducing background in mass spectrometry by employing the modified streptavidin molecules.

Description

technical field [0001] The present invention relates to modified streptavidin molecules resistant to Lys-C or other protease cleavage. These modified streptavidin molecules can be prepared by chemical modification of natural streptavidin, by chemical synthesis or by genetic engineering. The invention also relates to nucleic acid molecules encoding these modified streptavidin molecules, to vectors comprising these nucleic acid molecules, and to cells comprising these nucleic acid molecules or vectors. The present invention further relates to solid phase supports and kits comprising said modified streptavidin molecules. The present invention also relates to the role of these modified streptavidin molecules or these solid phase supports in capturing / immobilizing proteins, peptides, oligonucleotides (eg aptamers), polynucleotides (DNA, RNA or PNA), Uses in lipids, (poly)saccharides, carbohydrates, metabolites, pharmaceuticals and small molecules, natural and synthetic molecules,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C07K14/36
CPCC07K14/36C12N9/64C12N9/50G01N33/5308G01N2440/32
Inventor 杰伦·克里伊格斯菲尔德穆罕默德-雷扎·拉菲
Owner EURO LAB FUER MOLEKULARBIOLOGIE EMBL
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