Recombinant acetylated lysine arginine N-terminal protease and preparing method and applications thereof

A technology for acetylating lysine-arginine and lysine-arginine, which is applied in the field of protease, can solve the problems of increasing the stability of protease at the N-terminal of lysine-arginine, and achieve high stability and high enzyme activity Effect

Active Publication Date: 2019-04-19
BEIJING PROTEOME RES CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no modification method to incre...

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  • Recombinant acetylated lysine arginine N-terminal protease and preparing method and applications thereof
  • Recombinant acetylated lysine arginine N-terminal protease and preparing method and applications thereof
  • Recombinant acetylated lysine arginine N-terminal protease and preparing method and applications thereof

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Embodiment 1

[0061] Example 1 [Construction of recombinant lysine arginine N-terminal protease Escherichia coli expression vector]

[0062] The recombinant lysine-arginine N-terminal protease gene sequence of the present invention is derived from Methanosarcina acetivorans (GenBank No. AE010299), and the gene sequence is shown in SEQ ID NO.2. We introduced a 6×His tag and an enterokinase cleavage site DDDDK at its N-terminus (a total of 15 amino acids were added) to obtain an open reading frame (ORF) encoding 357 amino acids, which is the sequence shown in SEQ ID NO.3, which corresponds to The protein sequence of the gene is shown in SEQ ID NO.4, and the gene coding sequence and corresponding protein sequence are also shown in figure 1 middle. Insert the sequence shown in SEQ ID NO.3 between the Nco I / Xho I restriction sites of the pET28a vector (Novagen, product number 69864-3CN) to obtain a recombinant lysine-arginine N-terminal protease recombinant expression vector, named pET -Lysarg...

Embodiment 2

[0064] Example 2 [Fermentation and Thalline Collection of BL21-LysargiNase Strain]

[0065] The recombinant strain BL21-LysargiNase was activated on a solid LB / Amp plate to obtain a single clone of the BL21-LysargiNase strain. A single clone of BL21-LysargiNase strain was inoculated in 50 mL of LB liquid medium (the concentration of ampicillin was 100 μg / mL), and cultured on a shaker at 37°C. When the OD of LB liquid culture system 600 When it is 1.5-2.0 (with LB liquid medium as blank control), the seed liquid of BL21-LysargiNase bacterial strain is obtained.

[0066] The seed liquid of the BL21-LysargiNase strain was inoculated separately into a 14L fermenter (BioFlo 310, New Brunswick Scientific Company) equipped with 5L high-density fermentation basal medium sterilized by 121°C damp heat and autoclave, and the culture system after inoculation OD 600 0.01 (with the high-density fermentation basal medium as the blank control). Initial fermentation parameters: the culture...

Embodiment 3

[0074] Embodiment 3 [purification of recombinant lysine arginine N-terminal protease]

[0075] Get the BL21-LysargiNase thalline that 200g embodiment 2 obtains and resuspend in 20mM Tris pH 7.5 according to the ratio of 1:10 (w / v), 150mM NaCl damping fluid, mix homogeneously with high-speed tissue shearing machine, high-pressure homogenate is broken (APV -1000 Homogenizer Breaker, SPX FLOW, Inc.). Set the pressure of the high-pressure homogenate to 800 bar, break continuously for 3 times and then centrifuge at 13,000 g at 4°C for 30 min. Collect the supernatant and perform metal-chelate affinity chromatography on AKTA Purifier (GE healthcare) (HisTrapFF 5 mL prepacked column, GE healthcare, Cat. No. 17-5255-01).

[0076] The chromatographic column was pre-equilibrated with buffer A (20mM Tris, 150mM NaCl, pH 7.5) before sample loading, and the sample loading flow rate was 5mL / min. After sample loading, use 10 column volumes of buffer A and 10 column volumes of 5% buffer solu...

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Abstract

Recombinant acetylated lysine arginine N-terminal protease and a preparing method and applications thereof in proteomic research are provided. The recombinant acetylated lysine arginine N-terminal protease has a reduced self-digestion characteristic, improved stability and higher enzyme activity, can be used separately or combined with other protease, can improve the protein identification sequence cover degree, and the cover degree of readable amino acid residue sequences of trypsin digestion mirror peptide chain pairs and a peptide chain to be identified, and is a potential proteomic tool enzyme.

Description

technical field [0001] The invention belongs to the field of proteomics, and in particular relates to a protease used in proteomics research. [0002] The invention also relates to the preparation method of the protease and its application in proteomics research. Background technique [0003] The proteome is the sum of all proteins in a cell, tissue or organ, and is the final result of gene transcription, translation and post-translational modification. Studying the abundance, function, interaction, localization and regulation of proteins in cells is critical to our understanding of the mysteries of life. [0004] In the current most common proteomics research based on mass spectrometry, the identification of proteins and post-translational modifications requires enzymatic hydrolysis of protein samples into peptides, which are detected by mass spectrometry to obtain first-order spectra, and then peptide ions and inert gas Collisions are carried out in the mass spectrometer...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N9/96C12N15/70G01N33/68
CPCG01N33/6818C12N9/52C12N9/96C12N15/70
Inventor 徐平王富强张俊令赵明治常蕾
Owner BEIJING PROTEOME RES CENT
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