Method for improving biological functions of protein

A biological function and protein technology, which is applied in the field of improving the biological function of protein, can solve the problems of spending a lot of time and money, achieve good results, simple methods, and improve the biological function

Inactive Publication Date: 2006-04-26
NANJING UNIV OF TECH
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Problems solved by technology

This method of changing the chemical structure of existing drugs to find new

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  • Method for improving biological functions of protein
  • Method for improving biological functions of protein

Examples

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Embodiment 1

[0032] Embodiment 1: Transform the thermal stability of aspartate aminotransferase

[0033] 1. Using a rational design method, link the three-dimensional structure and one-dimensional structure of aspartate aminotransferase through sequence analysis software (Clustal W) and structure analysis software (Rasmol). On this basis, the protein structure prediction software (DeepView) was used to establish the functional region 1 and functional region 2 related to the thermostability of aspartate aminotransferase, which are two subunits (this enzyme consists of two identical subunits). base composition) between the two functional regions (functional region 1 is mainly the 5th to 12th amino acid of its N-terminus; functional region 2 is mainly its 277th to 294th amino acid). See figure 1 , where the arrow points to the modified region of the amplified three-dimensional structure of aspartate aminotransferase (that is, the functional regions 1 and 2 related to thermostability are regi...

Embodiment 2

[0059] Embodiment 2: Transform the substrate specificity of D-hydantoinase

[0060] In this example, the sites related to the substrate specificity of D-hydantoin enzyme were modified to improve its reaction to 5-monosubstituted hydantoin derivatives [D-5-benzylhydantoin (D-BH), D-5- P-hydroxybenzohydantoin (D-HBH), D-5-methylthioethylhydantoin (D-MTEH), D-5-isopropylhydantoin (D-IPH), D-5-methylhydantoin (D-MH)] substrate specificity.

[0061] 1. Using a rational design method, connect the three-dimensional structure and one-dimensional structure of Hydantoinase through docking experiment software (AutoDock) and molecular dynamics simulation software (GROMACS), and determine its sites directly related to substrate specificity For Tyr155, Ser288, Asp315, Asn336.

[0062] 2. The establishment of the mutant gene library includes the following steps:

[0063] 1) Introduction of random primers. In this experiment, a total of 12 bases were randomly mutated, that is, the capacit...

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Abstract

The present invention relates to method for raising biological function of protein. The method includes reasonable conventional protein design to determine the functional region related to the biological function of protein; substituting the gene in the functional region related to the biological function of protein with randomly changed primer segment and constituting mutant gene library in conventional gene engineering method; and the final directional protein evolution to screen out from the mutant gene library the protein with raised biological function. The method of the present invention can obtain protein with raised biological function, and is simple, practical and efficient.

Description

technical field [0001] The invention relates to a method for protein modification, in particular to a method for improving protein biological function. Background technique [0002] There have been many examples of directed evolution to solve industrial biocatalysts, but relatively speaking, rational design is rarely used in biocatalysts. In recent years, with the continuous advancement of nuclear magnetic resonance spectroscopy and X-ray crystallographic analysis, the ability of rational design will be developed rapidly, which will enable us to have a better understanding of protein folding, dynamics and structure-activity relationship. On the other hand, in directed evolution experiments, the number of all protein mutants is enormous for any experimental screening method. To overcome this limitation, structural calculation methods are commonly used to reduce the research time of directed evolution. For example, Voigt C A [Christopher A.Voigt, Stephen L.Mayo, Frances H.Ar...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/09G01N33/68G06F17/00
Inventor 严明许琳林涛欧阳平凯
Owner NANJING UNIV OF TECH
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