Optimized cattle chymosin proto-gene and secretory expression method and application thereof

Abstract

The invention discloses an optimized cattle chymosin proto-gene and a secretory expression method and the application thereof. According to the optimized cattle chymosin proto-gene, influence factors of difference between protein interpret rates and the like due to use frequency of codon, adjustment of GC content, deletion of unstable sequences, and different distribution of secondary mRNA structure and the codon are comprehensively considered, and optimized cattle chymosin proto-gene bodies respectively shown by SEQ ID NO.1, 2 and 3 are obtained by modifying cattle chymosin proto-gene bodies according to the preference codon of pichia pastoris. The invention further provides a production method of recombining the cattle chymosin proto-gene. The production method comprises the steps of converting recombinant expression carriers containing the optimized cattle chymosin proto-gene bodies into host cells to obtain recombination bacterial strains, cultivating the recombination bacterial strains, inducing expression of the recombination cattle chymosin proto-gene bodies, and recycling and purifying expressed products. Compared with original cattle chymosin proto-gene, the secretory expression amount and the enzyme activity of the optimized cattle chymosin proto-gene in the pichia pastoris are remarkably promoted.

Description

technical field

[0001] The present invention relates to an optimized prochymosin gene, in particular to a prochymosin gene derived from bovine prochymosin gene after codon optimization. The present invention also relates to an optimized prochymosin gene used in the production of The application in lactase belongs to the field of preparation of recombinant chymosin. Background technique

[0002] Cheese is rich in nutrition, delicious, long storage period and various in variety. It is an important part of the world's food and is known as the "king of dairy products". In the cheese production process, curd is a key process that affects the yield and quality of cheese. Chymosin (chymosin, EC3.4.23.4) is a key enzyme that plays a role in curdling in the process of curdling cheese. It is one of the aspartic acid proteases and can specifically crack k-casein Phe in 105- met 106 Peptide bonds, which cause milk protein coagulation, play an important role in dairy products, especi...

Examples

Embodiment 1

[0044] The optimization of embodiment 1 original gene of chymosin

[0045] 1.1 Optimization of the original gene of prorennin

[0046] The bovine prorennin gene pcw (GenBank Accession No. FJ768675.1) was selected as the original gene sequence. The prochymosin original gene (pcw) has a full length of 1098bp (SEQ ID NO.4), encoding 365 amino acids and a stop codon. Through the analysis of the sequence characteristics of the original gene sequence of bovine prochymosin, according to the principle of not changing the amino acid sequence of the protein, the frequency of codon usage, GC content, common enzyme cutting sites, yeast unstable sequences ATTTA, AACCAA, AATAA, translation The sequence of bovine prochymosin gene was preliminarily modified according to the codon preference of Pichia pastoris according to various influencing factors such as the secondary structure of mRNA in the initiation region. A total of 218 bases were changed, accounting for 19.8% of the total number o...

Embodiment 2

[0052] Example 2 Recombinant expression of bovine prochymosin in Pichia pastoris

[0053] 2.1 Experimental materials

[0054] 2.1.1 Strains and plasmids

[0055] Pichia pastoris GS115 strain and vector pPIC9 were purchased from Invitrogen; Escherichia coli Trans1-T1 competent was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; pGH-pcm, pGH-pcm-1, pGH-pcm-2 plasmids were purchased from Beijing Aoke Synthesized by Dingsheng Biotechnology Co., Ltd.

[0056] 2.1.2 Culture medium and related solution preparation

[0057] 1. LB medium: Dissolve 10g of peptone, 5g of yeast extract, and 10g of sodium chloride in a beaker, dilute to 1000ml with deionized water (the solid contains 15g of agar), sterilize with damp heat at 121°C for 20min.

[0058] 2. MD solid medium: Dissolve 2g of glucose and 2g of agarose in a beaker, dilute to 100ml with deionized water, and sterilize with damp heat at 108°C for 30min.

[0059] 3. BM mother liquor: 10g yeast extract, 20g peptone in 100...

Embodiment 3

[0088] Embodiment 3 Fermentation of rennet recombinant Pichia pastoris

[0089] Recombinant Pichia pastoris pcw-105 with the highest expression of chymosin from the original prorennin gene (pcw) # And the pcm-4 with the highest enzyme activity of the transoptimized prochymosin gene (pcm, pcm-1, pcm-2) which was significantly improved compared with the control strain in the re-screening experiment # , pcm1-220 # , pcm2-116 # A laboratory 3-liter fermenter was used to induce enzyme-producing fermentation.

[0090] Pick the transformants of the above four strains of yeast into 40ml YPD medium with a sterile toothpick, culture on a shaker (28°C, 200rpm) for 48h, then transfer to 200ml of YPD medium, culture on a shaker (28°C, 200rpm) 24h as the fermented seed bacterial liquid, inoculated in 3 liters of fermentation tanks. The parameters of the fermenter are set to pH 5.5, temperature 30°C, stirring rate 1000rpm, ventilation volume 200, and initial inoculum volume 200ml of bact...

technical field

[0001] The present invention relates to an optimized prochymosin gene, in particular to a prochymosin gene derived from bovine prochymosin gene after codon optimization. The present invention also relates to an optimized prochymosin gene used in the production of The application in lactase belongs to the field of preparation of recombinant chymosin. Background technique

[0002] Cheese is rich in nutrition, delicious, long storage period and various in variety. It is an important part of the world's food and is known as the "king of dairy products". In the cheese production process, curd is a key process that affects the yield and quality of cheese. Chymosin (chymosin, EC3.4.23.4) is a key enzyme that plays a role in curdling in the process of curdling cheese. It is one of the aspartic acid proteases and can specifically crack k-casein Phe in 105- met 106 Peptide bonds, which cause milk protein coagulation, play an important role in dairy products, especi...

Examples

Embodiment 1

[0044] The optimization of embodiment 1 original gene of chymosin

[0045] 1.1 Optimization of the original gene of prorennin

[0046] The bovine prorennin gene pcw (GenBank Accession No. FJ768675.1) was selected as the original gene sequence. The prochymosin original gene (pcw) has a full length of 1098bp (SEQ ID NO.4), encoding 365 amino acids and a stop codon. Through the analysis of the sequence characteristics of the original gene sequence of bovine prochymosin, according to the principle of not changing the amino acid sequence of the protein, the frequency of codon usage, GC content, common enzyme cutting sites, yeast unstable sequences ATTTA, AACCAA, AATAA, translation The sequence of bovine prochymosin gene was preliminarily modified according to the codon preference of Pichia pastoris according to various influencing factors such as the secondary structure of mRNA in the initiation region. A total of 218 bases were changed, accounting for 19.8% of the total number o...

Embodiment 2

[0052] Example 2 Recombinant expression of bovine prochymosin in Pichia pastoris

[0053] 2.1 Experimental materials

[0054] 2.1.1 Strains and plasmids

[0055] Pichia pastoris GS115 strain and vector pPIC9 were purchased from Invitrogen; Escherichia coli Trans1-T1 competent was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; pGH-pcm, pGH-pcm-1, pGH-pcm-2 plasmids were purchased from Beijing Aoke Synthesized by Dingsheng Biotechnology Co., Ltd.

[0056] 2.1.2 Culture medium and related solution preparation

[0057] 1. LB medium: Dissolve 10g of peptone, 5g of yeast extract, and 10g of sodium chloride in a beaker, dilute to 1000ml with deionized water (the solid contains 15g of agar), sterilize with damp heat at 121°C for 20min.

[0058] 2. MD solid medium: Dissolve 2g of glucose and 2g of agarose in a beaker, dilute to 100ml with deionized water, and sterilize with damp heat at 108°C for 30min.

[0059] 3. BM mother liquor: 10g yeast extract, 20g peptone in 100...

Embodiment 3

[0088] Embodiment 3 Fermentation of rennet recombinant Pichia pastoris

[0089] Recombinant Pichia pastoris pcw-105 with the highest expression of chymosin from the original prorennin gene (pcw) # And the pcm-4 with the highest enzyme activity of the transoptimized prochymosin gene (pcm, pcm-1, pcm-2) which was significantly improved compared with the control strain in the re-screening experiment # , pcm1-220 # , pcm2-116 # A laboratory 3-liter fermenter was used to induce enzyme-producing fermentation.

[0090] Pick the transformants of the above four strains of yeast into 40ml YPD medium with a sterile toothpick, culture on a shaker (28°C, 200rpm) for 48h, then transfer to 200ml of YPD medium, culture on a shaker (28°C, 200rpm) 24h as the fermented seed bacterial liquid, inoculated in 3 liters of fermentation tanks. The parameters of the fermenter are set to pH 5.5, temperature 30°C, stirring rate 1000rpm, ventilation volume 200, and initial inoculum volume 200ml of bact...

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