Streamlined Protein Expression and Purification Using Cleavable Self-Aggregating Tags.
Summary of "Streamlined Protein Expression and Purification Using Cleavable Self-Aggregating Tags."
Recombinant protein expression and purification remains a fundamental issue for biotechnology. Recently we found that two short self-assembling amphipathic peptides 18A (EWLKAFYEKVLEKLKELF) and ELK16 (LELELKLKLELELKLK) can induce the formation of active protein aggregates in Escherichia coli (E. coli), in which the target proteins retain high enzymatic activities. Here we further explore this finding to develop a novel, facile, matrix-free protein expression and purification approach.
In this paper, we describe a streamlined protein expression and purification approach by using cleavable self-aggregating tags comprising of one amphipathic peptides (18A or ELK16) and an intein molecule. In such a scheme, a target protein is first expressed as active protein aggregate, separated by simple centrifugation, and then released into solution by intein-mediated cleavage. Three target proteins including lipase A, amadoriase II and beta-xylosidase were used to demonstrate the feasibility of this approach. All the target proteins released after cleavage are highly active and pure (over 90% in the case of intein-ELK16 fusions). The yields were in the range of 1.6-10.4 mug/mg wet cell pellet at small laboratory scale, which is comparable with the typical yields from the classical his-tag purification, the IMPACT-CN system (New England Biolabs, Beverly, MA), and the ELP tag purification scheme.
This tested single step purification is capable of producing proteins with high quantity and purity. It can greatly reduce the cost and time, and thus provides application potentials for both industrial scale up and laboratorial usage.
This article was published in the following journal.
Name: Microbial cell factories
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/21631955
- DOI: http://dx.doi.org/10.1186/1475-2859-10-42
Medical and Biotech [MESH] Definitions
Hepatocyte Nuclear Factor 3-gamma
A winged-helix transcription factor that regulates GENE expression in metabolic tissues. It plays a role in HOMEOSTASIS of GLUCOSE and controls expression of GLUT2 PROTEIN.
Nuclear Receptor Subfamily 1, Group D, Member 1
A DNA-binding orphan nuclear receptor that negatively regulates expression of ARNTL TRANSCRIPTION FACTORS and plays a role as a regulatory component of the circadian clock system. The Nr1d1 nuclear receptor expression is cyclically-regulated by a feedback loop involving its positive regulation by CLOCK PROTEIN; BMAL1 PROTEIN heterodimers and its negative regulation by CRYPTOCHROME and PERIOD PROTEINS.
Platelet Activating Factor
A phospholipid derivative formed by PLATELETS; BASOPHILS; NEUTROPHILS; MONOCYTES; and MACROPHAGES. It is a potent platelet aggregating agent and inducer of systemic anaphylactic symptoms, including HYPOTENSION; THROMBOCYTOPENIA; NEUTROPENIA; and BRONCHOCONSTRICTION.
Ribosomal Protein S6 Kinases
A family of protein serine/threonine kinases which act as intracellular signalling intermediates. Ribosomal protein S6 kinases are activated through phosphorylation in response to a variety of HORMONES and INTERCELLULAR SIGNALING PEPTIDES AND PROTEINS. Phosphorylation of RIBOSOMAL PROTEIN S6 by enzymes in this class results in increased expression of 5' top MRNAs. Although specific for RIBOSOMAL PROTEIN S6 members of this class of kinases can act on a number of substrates within the cell. The immunosuppressant SIROLIMUS inhibits the activation of ribosomal protein S6 kinases.
Y-box-binding Protein 1
Y-box-binding protein 1 was originally identified as a DNA-binding protein that interacts with Y-box PROMOTER REGIONS of MHC CLASS II GENES. It is a highly conserved transcription factor that regulates expression of a wide variety of GENES.
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