CN103320458A

CN103320458A - High efficient expression method for actinomyces-based nitrile hydratase gene in escherichia coli

Info

Publication number: CN103320458A
Application number: CN2012103451545A
Authority: CN
Inventors: 周哲敏; 余越春; 崔文璟; 周丽; 何琛辉
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2012-09-18
Filing date: 2012-09-18
Publication date: 2013-09-25
Anticipated expiration: 2032-09-18
Also published as: CN103320458B

Abstract

The invention discloses a high efficient expression method for an actinomyces-based nitrile hydratase gene in escherichia coli, and belongs to the field of microbial genetic engineering technology. The method processes high efficiency and safety. It is benefit for large scaled nitrile hydratase extraction and purification, and further theoretical research about nitrile hydratase that a large amount of soluble nitrile hydratase can be obtained in a short expression period by applications of the method.

Description

A kind ofly derive from actinomycetic Nitrile hydratase gene in the method for E. coli

Technical field:

A kind ofly derive from actinomycetic Nitrile hydratase gene in the method for E. coli, the present invention relates to a kind of in e. coli bl21 (DE3) method of successful expression Nitrile hydratase, belong to the microbiological genetic engineering field.

Background technology:

Nitrile hydratase (Nitrile hydratase is called for short NHase, EC 4.2.1.84) is a kind of metalloenzyme that the nitriles substance hydration can be become more valuable amides.NHase distributes more extensive in microorganism, can obtain at present the gene order of Nitrile hydratase at Rhod (Rhodococcus), Rhodopseudomonas (Pseudomonas), Pseudonocardia (Pseudonocardia), bacillus (Bacillus), Nocardia (Nocardia), Comamonas (Comamonas) middle part.

It is basic identical that the various countries investigator obtains the mode of Nitrile hydratase gene, general all is the genomic dna that extracts wild mushroom, then determine the gene order of Nitrile hydratase in this bacterial strain by consulting the range gene library, according to the primers at two ends, and obtain required cloned sequence with the method for polymerase chain reaction (PCR) amplification.Then commercial plasmid is connected enzyme with amplified fragments and cuts rear connection, construction recombination plasmid changes host strain over to again and expresses.

Be used at present expressing the Nitrile hydratase carrier and mainly contain pET21a (+), pET24a (+), pET28a (+), pBluescript II SK (+) and some shuttle plasmids.The host then take rhodococcus and intestinal bacteria as main, comprises rhodococcus DSM43985, rhodococcus ATCC12674, e. coli bl21, JM109, DH5 α and TG1 etc.

Because when the Nitrile hydratase gene in some actinomycetes sources is expressed in the system take intestinal bacteria as the host, suffered regulatory mechanism and wild mushroom More different, the albumen of expressing is very easily assembled the formation inclusion body, thus the heterogenous expression of the Nitrile hydratase gene in actinomycetes sources in the past in more than 10 year progress slow.

Kobayashi etc. are at the Nitrile hydratase of expression in escherichia coli rhodococcus rhodochrous J1, and expression level is extremely low, and it is more alive than enzyme only to be 0.012U/mg.The present invention be intended to develop a kind of can be in the method for the Nitrile hydratase in E. coli Rhod source.

Summary of the invention:

The method that the purpose of this invention is to provide a kind of Nitrile hydratase in E. coli Rhod source.

Described Nitrile hydratase gene BAE sees the Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1 such as NAGASAWA for details.Eur.J.Bio-chem。196(1991)581-589。

Described encoding nitrile hydratase gene nucleotide series is shown in SEQ ID NO.1.

Concrete steps of the present invention are as follows:

1) according to Nitrile hydratase original series design primer (BAE up, BAE down), enzyme is cut (Nde I Hind III) and is connected to carrier pET24a (+) behind the amplification total length Nitrile hydratase gene BAE, gives Shanghai to give birth to worker bio-engineering corporation and checks order.

2) design has partly overlapping upstream primer (SD2) with B gene downstream and A upstream region of gene respectively with intestinal bacteria SD sequence (AAGGAG), intervening sequence (ATATACAT) and two ends, design simultaneously with intestinal bacteria SD sequence, intervening sequence and two ends and have partly overlapping downstream primer (SD3) with A gene downstream and E upstream region of gene respectively, the B upstream region of gene use on pET24a (+) carrier self with SD sequence (shown in SEQ ID 6) and intervening sequence (shown in SEQ ID 7);

3) adopt designed pair of primers in the previous step, constructed pET24a (+) in the step 1-BAE carries out full plasmid amplification as template, inserts and replace BAE gene primary leading sequence;

4) full plasmid PCR product pET24a (+)-(rbs) B (rbs) A (rbs) E that obtains in the previous step is cut 4h with 37 ℃ of enzymes of Dpn I, guarantee that primary template pET24a (+)-BAE is cut, then change and turn e. coli jm109, coating contains dull and stereotyped rear 37 ℃ of cultivations of resistance;

5) picking list bacterium colony goes to the liquid LB culture medium culturing that contains resistance, extracts plasmid and carries out the nucleic acid electrophoresis checking, and enzyme is cut checking and PCR checking, then gives Shanghai to give birth to worker bio-engineering corporation and checks order;

6) recombinant plasmid that sequence is correct transforms BL21 (DE3), carries out abduction delivering, and SDS-PAGE detects the protein expression situation, and HPLC detects enzyme activity.

Nitrile hydratase gene source of the present invention belongs to actinomycetic a kind of in rhodococcus (Rhodococcus rhodochrous J1).The ORF of correct identification Nitrile hydratase gene order adopts the method for full plasmid amplification to insert colibacillary SD sequence (AAGGAG) and intervening sequence (ATATACAT) in Nitrile hydratase B gene, A gene and E gene order upstream.The PCR product is changed to turn in the e. coli bl21 (DE3) and is carried out abduction delivering after primary template pET24a (+)-BAE is removed in Dpn I digestion.

Beneficial effect of the present invention: the invention provides a kind of method of the Nitrile hydratase in E. coli Rhod source, the Nitrile hydratase gene that the method can the successful expression actinomycetes be originated in e. coli bl21 (DE3).

Description of drawings:

Fig. 1. the full gene PCR of Nitrile hydratase

1, dna molecular amount standard; 2, the full gene PCR of Nitrile hydratase

Fig. 2. the full plasmid PCR of Nitrile hydratase inserts intestinal bacteria SD sequence and intervening sequence

1, dna molecular amount standard; 2, (B, A, E upstream sequence replace to SD sequence (shown in SEQ ID 6) and the intervening sequence (shown in SEQ ID 7) in the intestinal bacteria to the full plasmid PCR product of Nitrile hydratase

Fig. 3. the SDS-PAGE electrophorogram that Nitrile hydratase is expressed

1, molecular weight of albumen standard; 2, pET24a (+)-(rbs) B (rbs) A (rbs) E cytoclasis supernatant.

Embodiment:

Material and detection method

The LB substratum: 1% Tryptones, 0.5% yeast extract, 1% sodium-chlor, pH 7.0.

TB substratum: 1.2% Tryptones, 2.4% yeast extract, 0.4% glycerine, 17Mm KH ₂PO ₄, 72mM K ₂HPO ₄

Nitrile hydratase HPLC testing conditions: moving phase phosphoric acid acetonitrile damping fluid; Detect wavelength 210nm; Chromatographic column is the C18 post.

Embodiment 1

1) PCR obtains the Nitrile hydratase full-length gene

By consulting Nitrile hydratase gene order design primer (BAE up, BAE down) amplification total length Nitrile hydratase gene order in the rhodococcus.

Upstream primer BAE up (containing Nde I restriction enzyme site) is shown in SEQ ID NO.2, and downstream primer BAE down (containing the HindIII restriction enzyme site) is shown in SEQ ID NO.3;

2) design has the pair of primers of intestinal bacteria SD sequence and intervening sequence

Express natural Nitrile hydratase complete sequence in the rhodococcus in the e. coli bl21, much more report is arranged in the world, but all undesirable, replace to colibacillary SD sequence (shown in SEQ ID 6) and intervening sequence (shown in SEQ ID 7) by the upstream intervening sequence with Nitrile hydratase α gene and modulin e gene, but a large amount of soluble Nitrile hydratases of successful expression.

SD1 (the SD sequence and the intervening sequence that contain the A upstream region of gene) is shown in SEQ ID NO.4, and SD2 (containing SD sequence and intervening sequence on the B gene) is shown in SEQ ID NO.5.

3) full plasmid PCR method is inserted and is replaced the SD sequence;

By full plasmid PCR method, can one the step intervening sequence that Nitrile hydratase is original replace to Nitrile hydratase gene with intestinal bacteria SD sequence;

4) with full plasmid PCR product in the previous step, after Dpn I processing, be cloned into pET24a (+) and transform abduction delivering in the e. coli bl21 (DE3);

5) the Nitrile hydratase enzyme was lived after HPLC detected and transforms;

Nitrile hydratase HPLC testing conditions: moving phase phosphoric acid acetonitrile damping fluid; Detect wavelength 210nm; Chromatographic column adopting C18 post.

Claims

1. method that derives from actinomycetic Nitrile hydratase gene high efficient expression in e. coli bl21 (DE3), to adopt in Nitrile hydratase B gene order, the A gene order, and modulin E gene order upstream replaces to colibacillary SD sequence (shown in SEQ ID 6) and intervening sequence (shown in SEQ ID 7), structure transforms in the e. coli bl21 (DE3) and carries out abduction delivering with pET24a-(rbs) B (rbs) A (rbs) E of colibacillary SD sequence (shown in SEQ ID 6) and intervening sequence (shown in SEQ ID 7).

2. the method for claim 1 is characterized in that, described Nitrile hydratase gene order derives from the rhodococcus (Rhodococcus rhodochrous J 1) in the actinomycetes.

3. the method for claim 1, it is characterized in that, express in e. coli bl21 (DE3) after replacing to colibacillary SD sequence (shown in SEQ ID 6) and intervening sequence (shown in SEQ ID 7) described Nitrile hydratase gene B, A upstream and regulatory protein gene E upstream.

4. the method for claim 1 is characterized in that, makes up pET24a (+)-(rbs) B (rbs) A (rbs) E expression vector.

5. the method for claim 1 is characterized in that, described expressive host bacterium is e. coli bl21 (DE3).

6. the method for claim 1 is characterized in that, described fermention medium is TB substratum (1.2% Tryptones, 2.4% yeast extract, 0.4% glycerine, 17mM KH ₂PO4,72mM K ₂HPO4) contain in addition 4% dehydrated alcohol.

7. the method for claim 1 is characterized in that, step is as follows:

1) according to Nitrile hydratase original series design primer, enzyme is cut and is connected to carrier pET24a (+), order-checking behind the amplification total length Nitrile hydratase gene.

2) two primers with intestinal bacteria SD sequence and intervening sequence that are used for full plasmid amplification of design;

3) adopt designed pair of primers in the previous step, constructed pET24a (+) in the step 1-BAE carries out full plasmid amplification as template;

4) full plasmid PCR product pET24a (+)-(rbs) B (rbs) A (rbs) E that obtains in the previous step is processed with Dpn I, transform in the e. coli jm109 and increase;

5) later order-checking of recombinant bacterium upgrading grain checking;

6) the correct recombinant plasmid of Selective sequence changes BL21 (DE3) over to and carries out abduction delivering.