High secretion expression of recombination thymosin-alpha 1 in Escherichia coli and separation and purification therefor

A technique of secreting and expressing Escherichia coli, which is applied in the field of bioengineering and can solve problems such as cumbersome and complex production processes

Active Publication Date: 2006-09-13
上海普洛创智医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the molecular weight of the polypeptide is small, it is easy to be hydrolyzed by protease when expressed in the host bacteria. It is reported that the level of direct expression can only reach the level of several milligrams per liter. Multi-copy expression of genes or expression of polypeptides in the form of fusion proteins, but the products expressed by these methods need to undergo post-processing steps such as chemical cleavage or enzyme digestion, and the production process is very complicated and cumbersome

Method used

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  • High secretion expression of recombination thymosin-alpha 1 in Escherichia coli and separation and purification therefor
  • High secretion expression of recombination thymosin-alpha 1 in Escherichia coli and separation and purification therefor
  • High secretion expression of recombination thymosin-alpha 1 in Escherichia coli and separation and purification therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Design and synthesis of recombinant thymosin α1 gene:

[0044] We designed the following two fragments (SEQ ID NO.3 and SEQ ID NO.4 in the sequence listing), the underlined part is the complementary region of the two fragments. When designing the synthetic fragments, the following principles were fully followed: according to the amino acid sequence of natural thymosin α1 (SEQ ID NO.10 in the sequence listing), the preferred codons of Escherichia coli were selected; the AT and GC contents were close and evenly distributed; the fragments To avoid the generation of secondary structure within; to avoid repeated sequences between fragments; to add two stop codons at the 3' end of the gene.

[0045] Fragment 1: 5′-TCTGACGCTGCTGTTGACACTTCTTCCGAAATCACTAC

[0046] CAAA GACCTGAAAGAAAAG -3′

[0047] Fragment 2: 5′-CCCAAGCTTATTAGTTTTCAGCCTCTTCTACAACTTCTTT

[0048] CTTTTCTTTCAGGTC -3′

[0049] Each of the two chemically synthesized gene fragments was 10D, ...

Embodiment 2

[0050] Example 2 Chemical synthesis of alkaline phosphatase phoA promoter and signal peptide gene:

[0051] We synthesized the gene completely according to the natural gene sequence (SEQ ID NO.5 in the sequence listing) of Escherichia coli alkaline phosphatase phoA promoter (phoA-P) and signal peptide (sig). The following four fragments (fragments 3-6) were first synthesized (SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 in the sequence listing), and the underlined part is the complementary region between the fragments. Add PstI, BglII, and XbaI restriction sites at the 5' end of phoA-P to facilitate construction.

[0052] Fragment 3: 5′-AACTGCAGATCTAGAGCTCGTCAGTAAAAAGTTAATCTTT

[0053] TC AACAGCTGTCATAAAGTT -3'

[0054] Fragment 4: 5'- TTAAAAAATAAAAACAAA GCGACTATAAGTCTCGGCCGTG

[0055] AC AACTTTATGACAGCTGTT -3'

[0056] Fragment 5: 5'- TTTGTTTTTTATTTTTAA TGTATTTGTACATGGAGAAAAT

[0057] AAA AGTGAAAACAAAGCACTAT -3'

[005...

Embodiment 3

[0060] Example 3 Construction of expression plasmids ( figure 1 ):

[0061] 3.1 Enzyme digestion treatment of vector:

[0062] Plasmid pTZ18R (Pharmacia Company) was used as a starting plasmid. Take 5 μg of pTZ18R, add 20 UPstI enzyme (TaKaRa company) and 10 μl 10×PstI enzyme buffer (TaKaRa company), add distilled water to 100 μl, digest at 37°C for 4 hours, then add 10 μl 3M sodium acetate, 200 μl absolute ethanol, fully Mix well, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 200 μl of 70% ethanol, mix well, centrifuge at 12000rpm for 2 minutes, discard the supernatant. After the precipitate was dried, add 20U HindIII enzyme (TaKaRa Company) and 5 μl 10×HindIII enzyme buffer (TaKaRa Company), supplement distilled water to 50 μl, digest overnight at 37°C, and electrophoresis the digestion mixture in 1% agarose gel. Cut out a band of about 2.9 kb, use the DNA gel recovery kit (Shanghai Sangon Bioengineering Technology Service Co., Ltd.), recover the DNA...

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Abstract

The invention supplies a recombination thymosin alpha 1 gene, expression particle pKAT, engineering bacterium YKAT containing pKAT and filtered engineering strain YKAT-8, and the manufacture method for recombination thymosin alpha 1. It uses colibacillus preference codon, designing and combining recombination thymosin alpha 1 gene, recombining the upstream sequence, constructing pKAT and constructing and filtering YKAT-8. After fermenting and cultivating, directly excreting rT alpha-1 into culture medium, and the excreting quality could reach 500mg / L, and the purity could be over 95% after separating and purifying. It could sharply decrease producing cost and production cycle.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to the preparation of recombinant thymosin α1 through a genetic engineering method. Background technique [0002] The thymus is the most important immune organ of the human body and plays a key role in the immune system. In the mid-1970s, people isolated a mixed thymus polypeptide preparation from thymus tissue, called TF5, and the TF5 component was considered to be A powerful immune system enhancer, which can promote the differentiation and maturation of T cells and their normal functions, increase the body's antibody expression and immune transplantation response, and promote the expression of MIF (macrophage migration inhibitory factor) and so on. [0003] Thymosin α1 (Thymosin α1, referred to as: Tα1) is the first active polypeptide purified from TF5 fraction to obtain a homogeneous state. It consists of 28 amino acids, the N-terminus is acetylated, the isoelectric poin...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N1/21C12N15/70C12P21/02C07K1/14
Inventor 甘人宝张倩冯宝山钱悦杜鹏丁红珍周庆玮
Owner 上海普洛创智医药科技有限公司
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