Starch binding domain and use thereof

A starch and region technology, applied in the direction of introducing foreign genetic material, peptides, enzymes, etc. using vectors, can solve the problems of inconvenient purification process, expensive and laborious purification columns, etc.

Inactive Publication Date: 2009-12-16
SIMPSON BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Disadvantages of these current protein purification systems include: their purification process is inconvenient and laborious, and the columns used for purification are expensive

Method used

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  • Starch binding domain and use thereof
  • Starch binding domain and use thereof
  • Starch binding domain and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (A) Construction of plasmid

[0050] The construction of the recombinant vector is as follows figure 1 shown. The eGFP, linker and SBD fragments were all amplified by PCR with the designed primers, and the primer sequences are shown in Table 1. The linker sequence can be replaced by the following five candidate sequences (RoLK: linker of Rhizopus oryzae GA, PH: six histidines, PK: eight lysines, PPT: one threonine and four proline-threo Amino acid repeat sequence [T(PT) 4 ] and 58L: the region between the restriction sites SpeI and NcoI on the vector pET39b(+). The procedure of the PCR reaction was as follows: mix 10 ng template, 0.5 μl of each primer (10 μM), 5 μl of reaction buffer (10×), 5 μl of deoxynucleotide (2.5 mM), 0.8 μl of Ex Taq DNA polymerase (Takara Mirus Bio, Japan , 5U / μl), add water to a final volume of 50 μl. This mixture reacts according to the following cycles: 1 cycle of 95°C / 5 minutes → 30 cycles of [95°C / 30 seconds (high temperature denaturat...

Embodiment 2

[0079] (A) Effect of pH value on adsorption capacity

[0080] In the experiment of testing the effect of pH value on the adsorption capacity, the purified fusion protein (16 μM) was stirred into cornstarch buffer (Sigma-Aldrich, EC 232 -679-6, USA) for about 1 hour. Binding assay at pH 2.0-11.0 buffer [100mM glycine / hydrochloric acid (pH 2-3), 100mM sodium acetate / acetic acid (pH 4-5), 100mM Na 2 HPO 4 / NaH 2 PO 4 (pH 6-7), 100mM Tris / HCl (pH 8), 100mM glycine / sodium hydroxide (pH 9-11)]. The concentration of the unadsorbed fusion protein in the supernatant before and after adsorption was determined by bicinchoninic acid reagent set (BCA assay). The relative adsorption capacity of 100% fusion protein is based on the measured value at pH 5.0.

[0081] (B) Effect of temperature on adsorption capacity

[0082] In the experiment of testing the effect of temperature on the adsorption capacity of starch, the purified fusion protein (16 μM) was stirred into the adsorption buff...

Embodiment 3

[0095] (A) Structure determination by NMR spectroscopy

[0096] NMR data were analyzed with a Bruker Avance 600MHz or 800MHz spectrometer. For structure determination, 1 mM RoCBM21 (unlabeled, 15 N-flag or 13 C, 15 N-double label) was dissolved in 10 mM sodium acetate, pH 4.5, for NMR experiments at 25°C. Protein concentration was quantified with bicinchoninic acid reagent kit (Bio-Rad Protein Assay). Skeleton assignments were performed with HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HNCO and HN(CA)CO experiments (Cavanagh, J. et al., (1996) Protein NMR spectroscopy, Academic Press Inc .). Since RoCBM21 contains a relatively high proportion of aromatic residues, the assignment of its aromatic branch was assisted by HBCBCGCDHD and HBCBCGCDCEHE experiments (Yamazaki, T. et al., (1993) J.Am.Chem.Soc.115, 11054 -11055). The remaining atoms are assigned by homonuclear two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and 15 N heteronuclear single-quantum coherenc...

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Abstract

The present invention relates to a starch binding domain, a recombinant protein and a complex thereof. The present invention also relates to a method for separating a recombinant protein comprising a starch binding domain of the present invention.

Description

technical field [0001] The present invention relates to starch adsorption region, recombinant protein and composition thereof. The present invention also relates to methods for isolating recombinant proteins comprising starch adsorption domains. Background technique [0002] The use of microbial systems to express proteins has become the main source of high-priced and important pharmaceutical proteins, and how to purify and recover recombinant proteins is an important consideration in the design of fermentation processes. Traditional protein purification methods can be used to purify a single product, and improved methods further include the use of recombinant proteins. The recombinant protein can be purified by affinity column chromatography, and the components of the recombinant protein to be purified can be purified by forming a covalent link with the polypeptide bound to the affinity matrix. [0003] Certain systems separate proteins by the principle of affinity column...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C07K1/22C12P19/14C12N9/30C12N15/62C12N15/66
CPCC07K2319/20C12N9/2428C12N15/62
Inventor 张大慈吕平江孙玉珠许嘉钦
Owner SIMPSON BIOTECH CO LTD
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