Abstract
Previously we constructed a fusion protein based on GLP-1 and globular adiponectin but unfortunately its yield was low because it was mainly expressed as inclusion bodies. Herein to optimize the soluble expression of this fusion protein we tried several fusion tag systems. Fusion tags, including GST-, Trx- and MBP-tag, greatly improved the soluble expression of the fusion protein. However, these tag-fusion proteins were aggregation-prone as judged by Native PAGE and gel filtration chromatography, and this aggregation reduced the specificity of enterokinase-mediated enzyme cleavage which was essential to remove the fusion tags. To improve the specificity of protein cleavage, we employed on-column cleavage for downstream purification. Finally using optimized expression followed by on-column cleavage, we obtained the product fusion protein with a yield of 1.2 mg per g wet bacterial cells which was 8-fold higher than before. This method improved the yield and simplified the process, and as a convenient method it can also be used for the preparation of other aggregation-prone proteins.
Keywords: Adiponectin, enterokinase, fusion tag system, glucagon-like peptide-1, on-column cleavage, protein engineering, globular adiponectin, MBP-tag, gel filtration chromatography, enzyme.
Protein & Peptide Letters
Title:Optimized Soluble Expression and Purification of an Aggregation-prone Protein by Fusion Tag Systems and On-column Cleavage in Escherichia coli
Volume: 19 Issue: 12
Author(s): Wen Li, Mingming Gao, Wenchao Liu, Yuelin Kong, Hong Tian, Wenbing Yao and Xiangdong Gao
Affiliation:
Keywords: Adiponectin, enterokinase, fusion tag system, glucagon-like peptide-1, on-column cleavage, protein engineering, globular adiponectin, MBP-tag, gel filtration chromatography, enzyme.
Abstract: Previously we constructed a fusion protein based on GLP-1 and globular adiponectin but unfortunately its yield was low because it was mainly expressed as inclusion bodies. Herein to optimize the soluble expression of this fusion protein we tried several fusion tag systems. Fusion tags, including GST-, Trx- and MBP-tag, greatly improved the soluble expression of the fusion protein. However, these tag-fusion proteins were aggregation-prone as judged by Native PAGE and gel filtration chromatography, and this aggregation reduced the specificity of enterokinase-mediated enzyme cleavage which was essential to remove the fusion tags. To improve the specificity of protein cleavage, we employed on-column cleavage for downstream purification. Finally using optimized expression followed by on-column cleavage, we obtained the product fusion protein with a yield of 1.2 mg per g wet bacterial cells which was 8-fold higher than before. This method improved the yield and simplified the process, and as a convenient method it can also be used for the preparation of other aggregation-prone proteins.
Export Options
About this article
Cite this article as:
Li Wen, Gao Mingming, Liu Wenchao, Kong Yuelin, Tian Hong, Yao Wenbing and Gao Xiangdong, Optimized Soluble Expression and Purification of an Aggregation-prone Protein by Fusion Tag Systems and On-column Cleavage in Escherichia coli, Protein & Peptide Letters 2012; 19 (12) . https://dx.doi.org/10.2174/092986612803521710
DOI https://dx.doi.org/10.2174/092986612803521710 |
Print ISSN 0929-8665 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5305 |
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
Diabetes and Its Complications: Therapies Available, Anticipated and Aspired
Current Diabetes Reviews Synthesis and Investigating Hypoglycemic and Hypolipidemic Activities of Some Glibenclamide Analogues in Rats
Mini-Reviews in Medicinal Chemistry subject Index To Volume 2
Current Gene Therapy Role of Zinc and Lysosomal Enzymes in Type 2 Diabetes Mellitus with Periodontitis
Current Nutrition & Food Science Nutrition and Physical Activity on Hypertension: Implication of Current Evidence and Guidelines
Current Hypertension Reviews Oxidative Stress in the ICU
Current Nutrition & Food Science DNA Vaccines: A Mini Review
Recent Patents on DNA & Gene Sequences Anti-Inflammatory and Antioxidant Properties of a New Arylidene-Thiazolidinedione in Macrophages
Current Medicinal Chemistry Socioeconomic Position and Type 2 Diabetes Mellitus in Europe 1999- 2009: a Panorama of Inequalities
Current Diabetes Reviews Evaluation of the Antidiabetic Property of Capparis Ovata Desf. Var. Palaestina Zoh. Extracts Using In vivo and In vitro Approaches
Endocrine, Metabolic & Immune Disorders - Drug Targets Oxaliplapin and Capecitabine (XELOX) Based Chemotherapy in the Treatment of Metastatic Colorectal Cancer: The Right Choice in Elderly Patients
Anti-Cancer Agents in Medicinal Chemistry The Role of the Novel Adipocyte-Derived Protein Adiponectin in Human Disease: An Update
Mini-Reviews in Medicinal Chemistry Effects of Fatty Acids and Glycation on Drug Interactions with Human Serum Albumin
Current Metabolomics Phosphodiesterase Type 5 Inhibitors for the Management of Erectile Dysfunction: Preference and Adherence to Treatment
Current Pharmaceutical Design Editorial (Thematic Issue: Current Topics in Pharmacogenomics)
Recent Patents on Biotechnology Porphyromonas Gingivalis Antigenic Determinants - Potential Targets for the Vaccine Development against Periodontitis
Infectious Disorders - Drug Targets Editorial The Role of Mesenchymal Stem Cells in Bone Regeneration: Underlying Mechanisms and Systemic Regulatory Factors
Current Stem Cell Research & Therapy Contribution of Inflammation to Fat Redistribution and Metabolic Disturbances in HIV-1 Infected Patients
Current Pharmaceutical Design Uncovering the Diversification of Tissue Engineering on the Emergent Areas of Stem Cells, Nanotechnology and Biomaterials
Current Stem Cell Research & Therapy Experimental Diabetes Mellitus Down-Regulates Large-Conductance Ca2+- Activated K+ Channels in Cerebral Artery Smooth Muscle and Alters Functional Conductance
Current Neurovascular Research