CN102492697A - Method for cloning and expressing nitrilre hydratase regulatory protein - Google Patents

Abstract

The invention discloses a method for cloning and expressing a nitrile hydratase regulatory protein, which is to add a His-tag to the N-terminal of the nitrile hydratase regulatory protein gene sequence, connect it with the nitrile hydratase mature enzyme gene, and co-express it in Escherichia coli BL21, belonging to The field of gene cloning technology. The method of the invention is simple to operate and has high efficiency and success rate.

Description

A method for cloning and expressing nitrile hydratase regulatory protein

Technical field:

A method for cloning and expressing a nitrile hydratase regulatory protein, in particular to a method for expressing a nitrile hydratase regulatory protein in Escherichia coli BL21. the

Background technique:

Nitrile hydratase (NHase for short, EC 4.2.1.84) is a metalloenzyme that catalyzes the transformation of nitrile compounds into amido compounds. The acrylamide produced by this enzyme is nearly one million tons, accounting for one-third of the total acrylamide output. Compared with traditional chemical methods, biotechnology has the advantages of low cost, less energy consumption and less pollution. At present, in developed countries such as the United States, Japan, and France, this biotechnology is replacing traditional chemical methods. In my country, although the biotechnology of nitrile hydratase to synthesize acrylamide started late, it developed rapidly. According to statistics in 2008, acrylamide produced by biological methods in my country accounted for 41% of the total acrylamide output. Acrylamide is a widely used basic chemical raw material, which plays an important role in petroleum exploration, papermaking, decoration, etc. With the continuous expansion of the market, the demand for acrylamide is also increasing. In addition, this enzyme is also used in the production of nicotinamide. Niacinamide is a group B vitamin that participates in the metabolism of the body in the form of coenzyme I and coenzyme II, plays a very important role in human and animal metabolism, and is widely used in medicine, food additives and feed production. the

The wide application of nitrile hydratase has prompted scientists to study the synthesis mechanism and catalytic mechanism of this metalloenzyme. Nitrile hydratase can be divided into two types: iron-containing and cobalt-containing according to the different metal ions contained, and can be divided into four types according to the difference in gene structure. Although they catalyze the same type of chemical reactions, the synthesis modes of these enzymes are different, and these differences are highlighted in the different ways of metal ion uptake. The uptake mechanism of cobalt ions in the first and second types of nitrile hydratases has been clarified, but the third type has not been reported, and the relevant conclusions of the fourth type of nitrile hydratase (iron-containing type) are not clear. The key reason hindering ascertaining the uptake mechanism of the third type of cobalt ion is that there is no method for successfully cloning and expressing the regulatory protein of this type of nitrile hydratase (represented by Pseudomonas putida nitrile hydratase). The reason is that the report of the ORF part of the nitrile hydratase regulatory gene sequence in Pseudomonas putida is wrong, and even if the ORF of the regulatory protein is correctly identified, the regulatory protein is difficult to detect on SDS-PAGE. the

Invention content:

The invention provides an open reading frame for recognizing the regulatory protein of nitrile hydratase, the nucleotide sequence of which is SEQ ID NO.1. the

The present invention also provides a method for cloning and expressing the nitrile hydratase regulatory protein by using the above open reading frame. the

In order to solve the above problems, the following technical solutions are provided:

1) Download the GenBank sequence number U89363.1 from NCBI and identify SEQ ID NO.1;

2) Add SD sequence (AAGGAG) and His-tag sequence before the regulatory protein gene sequence;

3) Cloning the sequence constructed in the previous step in tandem into pET-24a(+) downstream of the nitrile hydratase mature enzyme, and introducing it into Escherichia coli BL21 to induce expression;

4) HPLC detection of modified nitrile hydratase enzyme activity. the

The nitrile hydratase gene of the present invention is derived from Pseudomonas putida NRRL-18668 ("A stereoselective cobalt-containing nitrole hydratase" published in Biochemistry in 1997). the

Detailed steps of the present invention are:

(1) Obtain the correct regulatory protein gene sequence and identify the correct ORF

The primers were designed to correctly amplify the mature enzyme gene sequence and the regulatory protein gene sequence by referring to the nitrile hydratase gene sequence in Pseudomonas putida. Because the ORF part of the nitrile hydratase regulatory gene sequence in Pseudomonas putida is currently reported incorrectly (the regulatory protein gene sequence has 3 possible ORFs), and the regulatory protein is difficult to detect on SDS-PAGE, so the correct Identifying the ORFs of regulatory proteins adds to the difficulty. By trying various ORFs and adding His-tag protection at the N-terminus of the regulatory protein, the ORF that successfully expressed the active regulatory protein was finally determined to be the correct regulatory protein ORF;

(2) Add His-tag to the N-terminus of the regulatory protein gene sequence

Escherichia coli BL21 expresses the complete sequence of the natural nitrile hydratase in Pseudomonas putida. There have been many reports in the world that the regulatory protein cannot be successfully expressed. By adding a His-tag to the N-terminal of the regulatory protein gene sequence, the regulatory protein can be successfully expressed ;

(3) Overlapped PCR tandem nitrile hydratase mature enzyme and regulatory protein gene sequence

The regulatory protein gene sequence with His-tag added to the N-terminal of nitrile hydratase is connected in series downstream of the mature enzyme gene sequence by overlapping PCR;

(4) Cloned into pET-24a(+) and introduced into Escherichia coli BL21 for expression

Cloning the full sequence of nitrile hydratase constructed in the previous step into pET-24a (+) into Escherichia coli BL21 to induce expression;

(5) HPLC detection of modified nitrile hydratase enzyme activity

Nitrile hydratase HPLC detection conditions: mobile phase acetonitrile phosphate buffer; detection wavelength 210nm; chromatographic column adopts C18 column;

(6) N-terminal sequencing of nitrile hydratase regulatory protein

Purify the expressed protein through a nickel column and sequence the N-terminus. The sequencing result (see Figure 3) is the sequence corresponding to the His-tag, and this His-tag is artificially added by us, which shows that the protein is regulated by nitrile hydratase The protein will lay a good foundation for further analyzing the metal ion uptake mechanism of nitrile hydratase and improving the industrial production process of nitrile hydratase. the

Beneficial effects of the present invention: the present invention provides a method for cloning and expressing the nitrile hydratase regulatory protein, which can successfully express the nitrile hydratase regulatory protein in Escherichia coli BL21. the

Description of drawings:

Figure 1. Nitrile hydratase whole gene PCR

1. Protein molecular weight standard; 2. Nitrile hydratase whole gene PCR (regulatory protein gene N-terminal plus His-tag). the

Figure 2. SDS-PAGE electrophoresis of nitrile hydratase expression

1. Protein molecular weight standard; 2. Whole cells (including the complete sequence of pET-24a(+) and nitrile hydratase gene, in which the N-terminal of the regulatory protein is added with His-tag). the

Figure 3. N-terminal sequencing map of nitrile hydratase regulatory protein after purification

Detailed ways:

Materials and Testing Methods

The strain is Pseudomonasputida NRRL-18668, and the related literature "A stereoselective cobalt-containing nitrole hydratase" was published in Biochemistry in 1997. the

Nitrile hydratase HPLC detection conditions: mobile phase acetonitrile phosphate buffer; detection wavelength 210nm; chromatographic column is C18 column. the

Example 1

1) Obtain the correct regulatory protein gene sequence and identify the correct ORF

The primers were designed to correctly amplify the mature enzyme gene sequence and the regulatory protein gene sequence by referring to the nitrile hydratase gene sequence in Pseudomonas putida. Because the ORF part of the nitrile hydratase regulatory gene sequence in Pseudomonas putida is currently reported incorrectly (the regulatory protein gene sequence has 3 possible ORFs), and the regulatory protein is difficult to detect on SDS-PAGE, so the correct Identifying the ORFs of regulatory proteins adds to the difficulty. By trying various ORFs and adding His-tag protection at the N-terminus of the regulatory protein, the ORF that successfully expressed the active regulatory protein was finally determined to be the correct regulatory protein ORF;

2) Add His-tag to the N-terminus of the regulatory protein gene sequence

Escherichia coli BL21 expresses the complete sequence of the natural nitrile hydratase in Pseudomonas putida. There have been many reports in the world that the regulatory protein cannot be successfully expressed. By adding a His-tag to the N-terminal of the regulatory protein gene sequence, the regulatory protein can be successfully expressed;

3) Overlapping PCR tandem nitrile hydratase mature enzyme and regulatory protein gene sequence

The regulatory protein gene sequence with His-tag added to the N-terminal of nitrile hydratase is connected in series downstream of the mature enzyme gene sequence by overlapping PCR;

4) Cloned into pET-24a(+) and introduced into Escherichia coli BL21 for expression

Cloning the full sequence of nitrile hydratase in the previous step into pET-24a (+) into Escherichia coli BL21 to induce expression;

5) HPLC detection of modified nitrile hydratase enzyme activity

Nitrile hydratase HPLC detection conditions: mobile phase acetonitrile phosphate buffer; detection wavelength 210nm; chromatographic column adopts C18 column;

6) N-terminal sequencing of nitrile hydratase regulatory protein

Purify the expressed protein through a nickel column and sequence the N-terminus. The sequencing result (see Figure 3) is the sequence corresponding to the His-tag, and this His-tag is artificially added by us, which shows that the protein is regulated by nitrile hydratase The protein will lay a good foundation for further analyzing the metal ion uptake mechanism of nitrile hydratase and improving the industrial production process of nitrile hydratase. the

Claims (6)

1. an ORFs of discerning the Nitrile hydratase modulin is characterized in that, its nucleotides sequence is classified SEQ ID NO.1 as.

2. the method for a clonal expression Nitrile hydratase modulin is to add SD sequence and His-tag at the said ORFs front end of claim 1, connects coexpression in e. coli bl21 with Nitrile hydratase maturing enzyme gene.

3. method as claimed in claim 2 is characterized in that, said Nitrile hydratase gene order derives from pseudomonas putida (Pseudomonas putida) NRRL-18668.

4. method as claimed in claim 2 is characterized in that, makes up pUC19-AB (his-tag) P cloning vector and pET-24a (+)-AB (his-tag) P expression vector.

5. method as claimed in claim 2 is characterized in that, said expressive host bacterium is an e. coli bl21.

6. like the described arbitrary method of claim 2-5, it is characterized in that the recombination bacillus coli fermention medium of said expression Nitrile hydratase modulin is: 1.2% Tryptones, 2.4% yeast extract, 0.4% glycerine, 17mMKH ₂PO4,72mMK ₂HPO4.

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