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Escherichia coli for high expression of foreign protein and construction method and application thereof

A technology for Escherichia coli and exogenous protein, applied in the field of genetic engineering, can solve the problems of long stable period, difficult to maintain, and low yield of secreted exogenous protein.

Pending Publication Date: 2019-07-26
BIO THERA SOLUTIONS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

E.coli strain W3110 is a common host strain for expressing foreign proteins, but it is difficult to maintain a long stable period during high-density fermentation, and the yield of secreting foreign proteins is not too high

Method used

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  • Escherichia coli for high expression of foreign protein and construction method and application thereof
  • Escherichia coli for high expression of foreign protein and construction method and application thereof
  • Escherichia coli for high expression of foreign protein and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] 1. Transformation of BAT47

[0062] Escherichia coli BAT47 (E.coli strain W3110) was purchased from ATCC, and its genotype is: tonA ptr3△phoA△E15△(argF-lac)169degP41△ompT kan R .

[0063] For BAT47(tonA ptr3△phoA△E15△(argF-lac)169degP41△ompT kan R ) to carry out the transformation of spr and ilvG genes, the steps are as follows:

[0064] Step 1: mutate tryptophan (W, Trp) at position 174 encoded by the spr gene of the host bacterium into arginine (R, Arg) (the 520th bp T of the spr gene sequence is mutated into C).

[0065] 1. Construction of targeting vector pCVD442-spr

[0066] Primers were designed with reference to the spr gene (NCBI Locus tag: YP75_p2138) and its upstream and downstream sequences. Using BAT47 genomic DNA as a template, use spr-5F, spr-5R, spr-3F, and spr-3R primers (SEQ ID NO.1-4) with high-fidelity PCR enzymes to obtain full-length spr targeting fragments. Already contains T / C mutation, which can make tryptophan at position 174 of spr gene mu...

Embodiment 2

[0129] Example 2 Design and construction of foreign protein vector expressed in periplasmic space

[0130] The phoA promoter includes two parts, the core promoter (-40bp to +40bp) and the signal peptide. The schematic diagram of the phoA promoter structure is shown in Figure 10 . In order to effectively terminate the process of transcribing the exogenous protein sequence into RNA, a natural terminator such as T7 terminator or rrnB terminator can be used in the selection of the transcription terminator, but in order to make the transcription more effective termination, in this embodiment The engineered terminator λt0 was used (see Sequence No.). In order to enable the foreign protein to be correctly folded in the periplasmic space of E. coli, the sequence encoding the phoA promoter (SEQ ID NO.29) was fused with the foreign protein sequence and the transcription terminator λt0 (the structure diagram of the foreign protein is shown in Figure 11 shown), so that when the protei...

Embodiment 3

[0144] Example 3 Expression of Monoclonal Antibody Fragment Fab

[0145]In a 10L fermenter, the medium components are: casein hydrolysate (casein hydrolysate) 12g / L, sodium citrate dihydrate 1.2g / L, ammonium sulfate 6.2g / L, dipotassium hydrogen phosphate trihydrate 3.5g / L , sodium dihydrogen phosphate dihydrate 1.3g / L, defoamer 0.2ml / L (V / V), magnesium sulfate 1.44g / L, glucose monohydrate 3.36g / L, isoleucine 0.3g / L, three Ferric chloride hydrate 0.0337g / L, zinc sulfate heptahydrate 0.00575g / L, copper sulfate 0.00319g / L, boric acid 0.00124g / L, cobalt chloride hexahydrate 0.00476g / L, manganese sulfate monohydrate 0.00338g / L, Sodium molybdate dihydrate 0.00284g / L, kanamycin 0.05g / L, ampicillin 0.05g / L. Pick a single colony from the streaked LB plate, inoculate it into a 50ml centrifuge tube containing 12ml of LB medium (50μg / ml kanamycin, 50μg / ml ampicillin), and culture overnight at 37°C with shaking at 220rpm. About 19 hours. On the second day, take 10ml of the bacterial liq...

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Abstract

The invention provides an escherichia coli for high expression of a foreign protein. A genotype of the escherichia coli lacks a prc gene and carries a mutated spr gene and a mutated ilvG gene; the mutated spr gene is as follows: the 174th position encoded by a spr gene is mutated to arginine or lysine by tryptophan; and the mutated ilvG gene is as follows: two bases of 'TA' are inserted at the 981bp position of the ilvG gene. The escherichia coli achieves a stable period of growth of a escherichia coli strain in the high-density fermentation process, and the degradation of the foreign proteinsecreted and expressed in a periplasmic space of the escherichia coli is inhibited, the high expression of the foreign protein is achieved, and the expression level is improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a method for constructing Escherichia coli for highly expressing foreign proteins and its application. Background technique [0002] Escherichia coli (E.coli) can be used as a unique system for exogenous proteins due to its own advantages, such as low cost, high yield, fast growth, high transformation efficiency, and the ability to obtain and purify the target protein in a short time. Analysis, and the final protein yield is higher. In contrast, mammalian cell systems for obtaining intact antibody molecules suffer from high production costs and long expression cycles. Therefore, Escherichia coli is the host of choice for protein expression in most scientific research and applications. However, E. coli still has some problems in expressing some proteins that contain complex disulfide bond structures or require post-translational modification. Due to the unique advantages of t...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/31C12N15/70C12N15/65C12N15/90C12R1/19
CPCC07K14/245C12N15/70C12N15/65C12N15/902Y02A50/30
Inventor 吴晓云黄俊杰李胜峰俞金泉
Owner BIO THERA SOLUTIONS
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