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T vector for screening solubility expression of protein and construction method and application thereof

A soluble and carrier technology, applied in the field of genetic engineering, can solve the problems of low fluorescence contrast, fluorescence retardation, low fluorescence display intensity, etc., and achieve the effect of convenient screening.

Inactive Publication Date: 2010-12-29
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, these reporter proteins have some shortcomings: first, the intracellular fluorescence intensity is not high, the fluorescence contrast is small, and fluorescence hysteresis occurs. For example, GFP colonies produce obvious fluorescence after 48 hours; Determined, it is related to the molecular weight, water solubility and expression of the upstream target protein

Method used

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  • T vector for screening solubility expression of protein and construction method and application thereof
  • T vector for screening solubility expression of protein and construction method and application thereof
  • T vector for screening solubility expression of protein and construction method and application thereof

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Embodiment Construction

[0032] 1. Verification of the correlation between C-terminal fusion α-peptide and protein water solubility

[0033] (1) Three mutants of maltose-binding protein (MBP) were constructed according to literature reports: folding-deficient type and two inclusion body MBP mutants (Betton JM, Hofnung M. Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. The Journal of Biological Chemistry.1996,271(14):8046-8052; Bajaj K, Chakrabarti P, VaradarajanR. Mutagenesis-based definitions and probes of residue burialin proteins.Proc Natl Acad Sci USA.2005,102(45):16221 -16226.).

[0034] (2) Construction of fusion expression vectors of C-terminal and α-peptide of wild-type and mutant MBP. The fusion expression vector of wild-type MBP and α-peptide is named MBP WT; the fusion expression vector of folding-deficient MBP and α-peptide is named G32D, and the glycine (G) at position 32 of maltose-binding protein (MBP) is mutated to natural Aspartic acid (...

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Abstract

The invention relates to a T vector for screening protein mutants with high water solubility / foldability and a construction method and application thereof in the field of directed evolution of protein. The T vector has the advantages of high throughput, high sensitivity and convenient screening. Six histidine (His) tags, multiple cloning sites (MCS) and the cobA gene sequence of coding red fluorescent protein UPMT are introduced into a departure vector pMAL-c2x so as to form the T vector. The T vector which is constructed by the method and used for screening the solubility expression of the protein is provided with a strong Ptac promotor, can directly clone, express and purify a PCR product, and particularly can conveniently screen the protein mutants with high water solubility / super foldability.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a T vector for high-throughput screening of soluble protein expression and a construction method thereof, and also relates to the application of the T vector in genetic engineering. Background technique [0002] PCR (Polymerase chain reaction) is a routine experimental technique in the field of molecular biology and genetic engineering research, and is the only way for gene cloning, gene or DNA fragment research. The most direct and convenient method for cloning PCR products is T / A cloning. The so-called T / A cloning is to clone the PCR product with a single A tail at the 3' end to a T vector with a single T tail at the 3' end. During PCR amplification, due to the template-independent terminal transferase activity of Taq enzyme, a single deoxynucleotide is added to the 3' end of the PCR product, and adenine deoxynucleotide is preferentially added among the four deoxynucleotides...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/65C07K2/00
Inventor 范军张宽亮魏玲玲
Owner ANHUI AGRICULTURAL UNIVERSITY
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