Shell nacre-like recombinant protein CSCa and method for regulating and preparing calcium carbonate by using same

A technology of recombinant protein and nacre, applied in the direction of calcium carbonate/strontium/barium, recombinant DNA technology, chemical instruments and methods, etc., can solve the problem of limited single protein, achieve low preparation cost, simple preparation method, understand mineralization Mechanism Facilitated Effects

Active Publication Date: 2016-03-30
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, single proteins are limited to specific stages of mineralization

Method used

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  • Shell nacre-like recombinant protein CSCa and method for regulating and preparing calcium carbonate by using same
  • Shell nacre-like recombinant protein CSCa and method for regulating and preparing calcium carbonate by using same
  • Shell nacre-like recombinant protein CSCa and method for regulating and preparing calcium carbonate by using same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Construction and expression of embodiment 1 recombinant protein CSCa

[0026] Experimental design The recombinant protein CSCa is composed of three fragments: chitin binding region, silk protein repeat sequence, and calcium ion binding region. The chitin-binding sequence is derived from the CBD sequence on the carrier plasmid PTWIN1, and the base sequence is shown in SEQ ID NO.4; the silk protein repeat sequence is derived from the silk protein of Bombyx mori, and the base sequence of the silk protein repeat sequence is shown in SEQ ID NO.5 ; The calcium ion binding sequence is derived from the calcium binding region of MS17 protein, and the base sequence is shown in SEQ ID NO.6.

[0027] First, the target gene fragment is amplified by PCR operation, and then the vector plasmid is digested, and then the vector plasmid and the PCR product are ligated overnight with DNA ligase, and finally the ligated product is transformed into competent cells, and the DNA electrophoresi...

Embodiment 2

[0029] Purification and dialysis of embodiment 2 recombinant protein CSCa

[0030]Wash 1.5ml chitin three times with LysisBuffer (Tris20mM, NaCl0.5M, EDTA1mM, Tween-201ml) and transfer to the column for use. After the collected cells were dissolved on ice, LysisBuffer (10mlLB / 100ml bacteria) was added in proportion, and then the cells were crushed three times with a high-pressure crusher. After centrifugation for 30 minutes (4°C, 12000 rpm / min), keep the supernatant. Add 5ml LB to the precipitate and leave it for protein electrophoresis. Pour the supernatant into the column to combine with chitin. The flow rate should be slow. Repeat the operation of the column to fully combine the protein and chitin. The total time for the column is about 4 hours. The process of the column is carried out in a low temperature environment , The supernatant after passing through the column needs to be placed on ice, and 30ul of the supernatant that passes through the chitin for the last time i...

Embodiment 3

[0032] Embodiment 3 recombinant protein CSCa regulates and prepares calcium carbonate

[0033] First wash 1-2ml of chitin with LysisBuffer (Tris 20mM, NaCl 0.5M, EDTA 1mM, Tween-201ml), mix the washed chitin with the recombinant protein CSCa solution, and combine overnight at 4°C. The next day, transfer the protein-bound chitin to a beaker and add NaHCO 3 Solution and deionized water, stir well and adjust to the desired pH, add CaCl 2 2H 2 O solution, where HCO 3 - ions and Ca 2+ The concentration of ions in the reaction system was 10mmol / L. After stirring for the designed time, the precipitate was collected by standing, washed with deionized water for 3 times, transferred to a glass dish, and freeze-dried to obtain a powder.

[0034] In this example, the effects of different protein contents, different mineralization time, and different mineralization initial pH on the mineralization results are designed.

[0035] (1) Protein content 2% (that is, 100ml mineralization re...

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Abstract

The invention specifically relates to a shell nacre-like recombinant protein CSCa and a method for regulating and preparing calcium carbonate by using the same, belonging to the field of material preparation. The recombinant protein CSCa is composed of a chitin binding domain, a silk protein repetitive sequence and a calcium ion binding domain. The method for regulating and preparing calcium carbonate by using the same comprises the following steps: uniformly mixing chitin with a recombinant protein CSCa solution; adding a NaHCO3 solution when chitin and recombinant protein CSCa are fully combined together and carrying out uniform mixing under stirring; then adjusting a pH value to 8.5 to 9.5 and adding a CaCl2.2H2O solution; and carrying out a mineralization reaction under the condition of stirring so as to obtain mineralized calcium carbonate. Vaterite-phase calcium carbonate can be prepared by using the method provided by the invention and can be stably maintained for a period of time under the action of the recombinant protein CSCa, which is an unusual phenomenon in the process of mineralization and is of great promotion significance to deeper understanding of the mechanism of mineralization.

Description

technical field [0001] The invention belongs to the field of material preparation, and in particular relates to a conchoid nacre recombinant protein CSCa and a method for preparing calcium carbonate through regulation and control thereof. Background technique [0002] As a typical natural biomineralized material, shell nacre has a special assembly method that is admirable to humans, so it has excellent mechanical properties such as high strength and toughness. The most prominent mechanical property of shell nacre is its high toughness, which is about 3000 times that of natural aragonite, mainly because the organic matrix plays an important role in the design of shell mechanical properties. The fundamental reason is that organisms use their own strong control over the construction of materials to regularly embed biopolymers in inorganic minerals or between mineral crystals through the modulation of cells. Although its proportion is small, it fundamentally changes the fractur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C01F11/18
CPCC01F11/183C01P2002/72C01P2004/03C07K14/43586C07K2319/00C12N15/62
Inventor 傅正义万亚敏谢浩王小莉
Owner WUHAN UNIV OF TECH
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