Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coli
Cowpea has a red nodule containing large amounts of bean hemoglobin. The protein is a good natural pigment and can be used as color agent in artificial meat. In this study, a 456 bp sequence of cowpea leghemoglobin Ⅱ (Lb II) was obtained from the NCBI database. Using the pET-15b as the expression ve...
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| Format: | Article |
| Language: | zho |
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The editorial department of Science and Technology of Food Industry
2023-02-01
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| Series: | Shipin gongye ke-ji |
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| Online Access: | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022050015 |
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| author | Meng LIU Congrui WANG Bo LIU Xiangzhong ZHAO |
| author_facet | Meng LIU Congrui WANG Bo LIU Xiangzhong ZHAO |
| author_sort | Meng LIU |
| collection | DOAJ |
| description | Cowpea has a red nodule containing large amounts of bean hemoglobin. The protein is a good natural pigment and can be used as color agent in artificial meat. In this study, a 456 bp sequence of cowpea leghemoglobin Ⅱ (Lb II) was obtained from the NCBI database. Using the pET-15b as the expression vector and E. coli BL21-CodonPlus (DE3)-R-IL as the recombinant expression host, the cowpea Lb Ⅱ was successfully expressed, then preliminarily purified this protein by using the nickel column chromatography and added ascorbic acid as the antioxidant simultaneously. IPTG concentration, temperature and time were used as the independent variables and Lb Ⅱ expression as the dependent variable to univariate experiments. The results showed that the highest yield of recombinant Lb Ⅱ protein was obtained at a final IPTG concentration of 1.0 mmol/L and 25 ℃ for 14 h. The recombinant protein was identified as Lb Ⅱ by SDS-PAGE and visible spectrophotometry methods. After the response surface experiment, the titer could reach 7.30 μg/mL in recombinant expression. This study would lay a basis for the subsequent fermentation and production of cowpea hemoglobin in other gene engineering strains. |
| first_indexed | 2024-04-10T16:39:06Z |
| format | Article |
| id | doaj.art-94be8b04e4004ada8124c73e7af69822 |
| institution | Directory Open Access Journal |
| issn | 1002-0306 |
| language | zho |
| last_indexed | 2024-04-10T16:39:06Z |
| publishDate | 2023-02-01 |
| publisher | The editorial department of Science and Technology of Food Industry |
| record_format | Article |
| series | Shipin gongye ke-ji |
| spelling | doaj.art-94be8b04e4004ada8124c73e7af698222023-02-08T08:00:16ZzhoThe editorial department of Science and Technology of Food IndustryShipin gongye ke-ji1002-03062023-02-0144416317010.13386/j.issn1002-0306.20220500152022050015-4Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coliMeng LIU0Congrui WANG1Bo LIU2Xiangzhong ZHAO3School of Food Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, ChinaSchool of Food Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, ChinaSchool of Food Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, ChinaSchool of Food Science and Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, ChinaCowpea has a red nodule containing large amounts of bean hemoglobin. The protein is a good natural pigment and can be used as color agent in artificial meat. In this study, a 456 bp sequence of cowpea leghemoglobin Ⅱ (Lb II) was obtained from the NCBI database. Using the pET-15b as the expression vector and E. coli BL21-CodonPlus (DE3)-R-IL as the recombinant expression host, the cowpea Lb Ⅱ was successfully expressed, then preliminarily purified this protein by using the nickel column chromatography and added ascorbic acid as the antioxidant simultaneously. IPTG concentration, temperature and time were used as the independent variables and Lb Ⅱ expression as the dependent variable to univariate experiments. The results showed that the highest yield of recombinant Lb Ⅱ protein was obtained at a final IPTG concentration of 1.0 mmol/L and 25 ℃ for 14 h. The recombinant protein was identified as Lb Ⅱ by SDS-PAGE and visible spectrophotometry methods. After the response surface experiment, the titer could reach 7.30 μg/mL in recombinant expression. This study would lay a basis for the subsequent fermentation and production of cowpea hemoglobin in other gene engineering strains.http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022050015cowpeahemoglobinresponse surface method optimizationrecombinant expression |
| spellingShingle | Meng LIU Congrui WANG Bo LIU Xiangzhong ZHAO Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coli Shipin gongye ke-ji cowpea hemoglobin response surface method optimization recombinant expression |
| title | Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coli |
| title_full | Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coli |
| title_fullStr | Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coli |
| title_full_unstemmed | Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coli |
| title_short | Optimization of Expression Conditions, Purification and Identification of the Recombinant Cowpea Hemoglobin Lb Ⅱ in Escherichia coli |
| title_sort | optimization of expression conditions purification and identification of the recombinant cowpea hemoglobin lb ii in escherichia coli |
| topic | cowpea hemoglobin response surface method optimization recombinant expression |
| url | http://www.spgykj.com/cn/article/doi/10.13386/j.issn1002-0306.2022050015 |
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