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Preparation method for beta-lactamase inhibitory polypeptide and expression vector used therein

A technology for inhibiting polypeptides and lactamases, which is applied in the fields of expression vectors, preparation of beta-lactamase inhibitory polypeptides, and fusion proteins, can solve the problems of high cost and difficulty in purification, and achieves low price, difficulty in purification and cost reduction. The ideal effect of enzyme digestion

Inactive Publication Date: 2012-05-09
SHANGHAI INST OF PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the technical problem to be solved in the present invention is to provide a new method for preparing a β-lactamase inhibitory polypeptide in view of the existing methods for preparing β-lactamase inhibitory polypeptides that are difficult to purify and high in cost. The method can greatly reduce the difficulty and cost of preparation and purification of β-lactamase inhibitory polypeptides

Method used

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  • Preparation method for beta-lactamase inhibitory polypeptide and expression vector used therein
  • Preparation method for beta-lactamase inhibitory polypeptide and expression vector used therein
  • Preparation method for beta-lactamase inhibitory polypeptide and expression vector used therein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction and protein expression of embodiment 1 expression vector

[0051] 1) Construction of pYG590

[0052] The TEV enzyme specifically recognizes the amino acid sequence of Glu-Asn-Lys-Tyr-Phe-Gln-Gly and cuts between Gln-Gly. The idea of ​​constructing the new vector is to combine the coding sequence of the recognition site of the TEV enzyme with the polypeptide P1 -A gene is connected and then cloned into the expression plasmid, the schematic diagram is shown in figure 1 .

[0053] Primers Primer5 and Primer6 with restriction sites for endonucleases BamHI and SalI were designed respectively (see Table 1). The polypeptide P1-A gene was amplified using pYG567 (see Chinese patent application 200910054340.1) as a template. The amplified product was double-digested with BamHI and SalI, and then combined with the pET32a-TEV plasmid (pET32a-TEV plasmid is the TEV recognition site cloned by the inventor of 7 amino acid sequences in pET32a, which was prepared The me...

Embodiment 2

[0080] The separation and purification of embodiment 2 small peptides

[0081] 1) TEV enzyme digests P1-A-TEV fusion protein

[0082] The purified P1-A-TEV fusion protein was appropriately concentrated to more than 1.0 mg / ml with a 10KDa ultrafiltration tube ultrafiltration (Amicon Ultra10K device, MILLIPORE Company), and the newly purified TEV enzyme in Example 1 was used according to the following table 2 The system shown in the enzyme cleavage reaction, 34 ° C water bath reaction 1-1.5h, the cleavage efficiency can exceed 50%.

[0083] Table 2. TEV enzyme digestion reaction system

[0084] 10× Enzyme Digestion Buffer

200μl

fusion protein

1.2-1.8mg

TEV enzyme

0.6-0.9mg

total capacity

2000μl

[0085] Among them, the 10× digestion buffer (1ml) is as follows:

[0086]

[0087] Using Tricine-SDS-PAGE electrophoresis detection, it can be seen that the P 1-A-TEV fusion protein is cut, and a small band with a size of about...

Embodiment 3P1

[0094] Example 3 In vivo inhibition experiment of P1-A-TEV fusion protein and polypeptide P1-A on drug-resistant bacteria

[0095] In order to detect whether the P1-A-TEV fusion protein obtained by the expression of the newly constructed vector pYG590 and the polypeptide P1-A have β-lactamase inhibitory activity, Klebsiella pneumoniae 10032 (ie ATCC700603) was used as the test bacteria for in vivo inhibition In the test, the β-lactamase inhibitor potassium clavulanate (purchased from Shanghai Xibao Biotechnology Co., Ltd., with a purity of 99.9%, meeting the standards of the United States Pharmacopoeia) was used as a control.

[0096] Firstly, the P1-A-TEV fusion protein purified from the broken supernatant, the P1-A-TEV fusion protein obtained by renaturation purification, the P1-A obtained by ultrafiltration and clavula were investigated as described in Example 2. Effect of potassium phosphate on the growth of Klebsiella pneumoniae. The P1-A-TEV fusion protein or polypeptid...

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Abstract

The invention discloses a preparation method for beta-lactamase inhibitory polypeptide and an expression vector, a fusion protein and the like used therein. The method comprises the following steps: 1) constructing a recombinant expression vector used for expressing His tag fusion protein which is a fusion protein of a TEV protease cleavage site His tag of beta-lactamase inhibitory polypeptide; 2) transforming host cells so as to obtain a transformant; 3) culturing the transformant and expressing the His tag fusion protein; 4) carrying out purification by using metal chelated affinity chromatography so as to obtain the His tag fusion protein; 5) carrying out restriction enzyme on the His tag fusion protein with TEV protease; 6) carrying out separation and purification by using gel column chromatography so as to obtain beta-lactamase inhibitory polypeptide. According to the invention, the effect of restriction enzyme of TEV protease is ideal, and TEV protease used in the method has a low price, thereby enabling both purification difficulty and cost of beta-lactamase inhibitory polypeptide to be substantially reduced.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, and in particular relates to a method for preparing a β-lactamase inhibitory polypeptide, an expression vector, a fusion protein and the like used therein. Background technique [0002] β-lactam antibiotics (including penicillins and cephalosporins) are clinically used to treat a variety of bacterial infections, but with the widespread use of antibiotics and abuse to some extent, drug-resistant bacteria are constantly emerging, and they Resistance to many β-lactam antibiotics, rendering such drugs ineffective against disease caused by resistant bacteria. Clavulanic acid, an inhibitor of microbial-derived β-lactamase, and its β-lactam sulfone compounds, Sulbactam and Tazobactam, were developed in the 1970s and 1980s. It was discovered and proved to be an effective β-lactamase inhibitor, and as a "suicide" combined with β-lactam antibiotics, it can achieve the purpose of inhibiting β-lactamas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/62C12N1/15C12N1/19C12N1/21C12N5/10C07K14/00C07K1/22C07K1/16C07K19/00A61K38/16A61P31/04
Inventor 胡又佳谢丽萍朱春宝朱宝泉许明飞
Owner SHANGHAI INST OF PHARMA IND
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