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Applications of beta-lactamase in improving functional protein folding and preparation of fusion protein

A technology of lactamase and fusion protein, applied in the field of fusion protein, can solve the problem that the leader sequence does not have a catalytic effect, achieve the effect of fewer steps and promote folding

Active Publication Date: 2012-12-05
TONGHUA DONGBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] d) The leader sequence does not have a catalytic effect, that is, one molecule of the leader sequence can only refold one molecule of the target protein;

Method used

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  • Applications of beta-lactamase in improving functional protein folding and preparation of fusion protein
  • Applications of beta-lactamase in improving functional protein folding and preparation of fusion protein
  • Applications of beta-lactamase in improving functional protein folding and preparation of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Synthesis of Encoding Fusion Protein SEQ ID No.5 DNA Sequence

[0031] (1) Gene design

[0032] Using computer-aided design, the structural genes are all selected codons preferred by Escherichia coli, designed according to the amino acid sequence of SEQ ID No.5, retrieved by PC-GENE software, and adjusted to individual codons to eliminate any direct and reverse repeat sequences and Inappropriate restriction site. At the 5'-end of the structural gene, a start codon ATG is added, and a stop codon TAA, TGA and a ribosome binding site suitable for complete protein expression are added at the 3'-end.

[0033] (2) Chemical synthesis of oligonucleotide fragments

[0034] The SEQ ID No.5 gene was divided into 30 oligonucleotide fragments of 30-40mers, which were respectively synthesized on an ABI381A DNA synthesizer by the solid-phase phosphite amine method. Then, end-labeling is performed and the purity of the fragments is checked by autoradiography.

[0035] (3)...

Embodiment 2

[0039] Example 2 Synthesis of Encoding Fusion Protein SEQ ID No.6 DNA Sequence

[0040] (1) Gene design

[0041] Using computer-aided design, the structural genes are all selected codons preferred by Escherichia coli, designed according to the amino acid sequence of SEQID No.6, retrieved by PC-GENE software, and adjusted to individual codons to eliminate any direct and reverse repeat sequences and non-existent sequences. Appropriate restriction sites. At the 5'-end of the structural gene, a start codon ATG is added, and a stop codon TAA, TGA and a ribosome binding site suitable for complete protein expression are added at the 3'-end.

[0042] (2) Chemical synthesis of oligonucleotide fragments

[0043] The gene of SEQ ID No.6 was divided into 10 oligonucleotide fragments of 28-35mers, which were respectively synthesized on the ABI381A DNA synthesizer by the solid-phase phosphite amine method. Then, end-labeling is performed and the purity of the fragments is checked by auto...

Embodiment 3

[0048] Preparation of Trypsinogen in the Existence of Example 3 Leader Sequence

[0049] (1) Acquisition of DNA and Construction of Bacterial Expression Vectors

[0050] A DNA fragment encoding the amino acid sequence of SEQ ID No.5 was chemically synthesized by the solid-phase phosphite amine method (see Example 1 for the specific method). The synthetic DNA fragment contains a fragment from 5' end Cla I to 3' end Kpn I and a fragment from 5' end Kpn I to 3' end Hind III, and they are respectively cloned into the pUC vector, wherein Kpn I can cut the coding Nucleic acid sequence of amino acid residues 51 and 52 of SEQ ID No.5. Then the DNA fragment encoding the complete amino acid sequence of SEQ ID No.5 was subcloned into the modified pATH2 vector, so that the expression of the fusion protein was regulated by the Trp promoter and the SD sequence. The obtained vector pTRY-1 is used for the expression of BL-trypsinogen fusion protein.

[0051] (2) Expression of BL-trypsinoge...

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Abstract

The invention provides applications of beta-lactamase or peptide of which the identity is at least 20% with a beta-lactamase amino acid sequence as leading peptide in improving functional protein folding. One or more of resectable amino acid residues in a leader sequence used in the invention are same with one or more resectable amino acid residues of an isolated leader sequence and a to-be-folded protein precursor, which is good for resecting leader sequence segments after folding, thus the purification steps at the down stream can be simplified, and also the leader protein provided by the invention can improve the folding of functional protein.

Description

technical field [0001] The present invention relates to a new application of β-lactamase, in particular to the new application of β-lactamase as a leading protein to promote the folding of functional protein, and the fusion protein prepared from the leading protein. Background of the invention [0002] The leader sequence of many proteins is essential for their correct folding and maturation. This leader sequence-dependent protein folding phenomenon was first discovered in 1987 by Ikemura et al. when studying subtilisin. Subtilisin is a kind of alkaline serine protease from Bacillus subtilis, the N-terminus of its zymogen is a leader sequence consisting of 77 amino acid residues. It is well documented that protein folding is regulated by a leader sequence in subtilisins. The "subtilisins" family includes enzymes of prokaryotic, eukaryotic and arcuate origin. The leader sequence is important for the production of active subtilisins both in vivo and in vitro, and as a leader...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/76C12N9/84C07K14/62C07K1/00C07K19/00
Inventor 冷春生
Owner TONGHUA DONGBAO PHARMA
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