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Luo D.,Sichuan University | Wen C.,Sichuan University | Zhao R.,Sichuan University | Liu X.,Sichuan University | And 4 more authors.
PLoS ONE | Year: 2016

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine-5′) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. © 2016 Luo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source


Ma Z.,Sichuan University | Luo D.,Sichuan University | Huang A.,University of Sichuan | Xu Y.,Sichuan University | And 6 more authors.
Gene | Year: 2014

The invention of DNA cloning over 40. years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes. © 2014 Elsevier B.V. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.80K | Year: 2008

DESCRIPTION (provided by applicant): Much efforts in gene expression analysis in the past have been focused mainly on the messenger RNAs (mRNAs), thanks to the availability of Differential Display (DD), SAGE and DNA microarray technologies, which all targ et the poly(A) tails present in most eukaryotic mRNAs. The recent discovery of a large microRNA population begged the question of whether there exists additional yet to be discovered RNAs species in a eukaryotic cell besides mRNA, rRNA and tRNA. However, i n contrast to the analysis of mRNA expression, analogous methods for an accurate, comprehensive detection and analysis of any nonpolyadenylated RNA have been lacking. Here we describe a systematic approach for the detection and identification of any non-po lyadenylated RNAs in a eukaryotic cell. The method involves first in vitro enzymatic addition of a poly(A) tail to all non-poly(A) RNAs in a cell followed by fluorescent Differential Display (FDD) comparison of cDNA patterns before and after poly(A) additi on. With the proof of principle established for the method, two well defined specific aims are formulated in this Phase I STTR application to further optimize and streamline the method for a more accurate and comprehensive screen for nonpolyadenylated RNA species expression in any eukaryotic cell. Specific Aim 1: Systematic Analysis of Non-poly(A) RNA Expression in Eukaryotic Cells by Differential Display a: Optimization of poly(A) tailing reaction of NPA-DD b: Poly(A) tailing of total RNA following the dep letion of poly(A) RNA and comparison of NPA-DD with tiling arrays, c: NPA-DD bypassing ribosomal RNA detection. Specific Aim 2: Comprehensive Test Screens for Non-poly(A) RNA Expression in Mammalian Cells.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.70K | Year: 2011

DESCRIPTION (provided by applicant): One of the modern strategies for treating autoimmune diseases such as rheumatoid arthritis and psoriasis involves the use of biologic TNF receptor decoys, such as soluble receptors or therapeutic antibodies, to intercept the inflammatory ligand TNF-1, and thus block the pathological activation of its receptors. However, current TNF-1 biologic blockers are all dimeric in structure, whereas TNF-1 itself is homotrimeric in nature. From a structural biology point of view, ahomodimeric structure with a two-fold symmetry cannot perfectly dock to a homotrimeric structure with a three-fold symmetry, thus limiting the affinity between the two molecules. Here we describe a general methodology for efficient creation of trimeric soluble receptors as secreted proteins. The process involves gene fusion between a soluble receptor with a ligand binding domain or any biologically active protein and a trimerization tag from the C-propeptide domain of pro-collagen (Trimer-Tag), which is capable of self-assembly into a disulfide bond-linked trimer. We show that the homotrimeric soluble TNF receptor produced with such method is a more potent blocker than dimeric TNF receptor decoys in inhibiting TNF- 1 signaling in vitro. Moreover, we have also demonstrated that covalently strengthened homotrimeric TRAIL/Apo2L-Trimer ligand is a potent anticancer agent, in contrast to its dimeric Fc fusion counterpart. Thus, Trimer-Tag has the potential to become a new platform technology for rational design ofthe next generation biologic drugs against autoimmune diseases, cancer, AIDS, osteoporosis, and heart disease. In this Phase I SBIR application, we seek to significantly increase the expression level and optimize the purification scheme of these recombinant trimeric fusion proteins in the hope that this novel technology, which is covered by 3 U.S. patents, can quickly move from preclinical stage towards the bedsides of millions of patients. PUBLIC HEALTH RELEVANCE: This Phase I SBIR application seeks to further optimize and streamline a newly patented protein trimerization technology for the design and production of secreted therapeutic biologics targeting major diseases such as autoimmune diseases, cancer, AIDS, osteoporosis, and heart disease.


Trademark
Genhunter Corporation | Date: 2005-03-15

Kit that contains research reagents for laboratory experiments for fluorescent gene expression analysis.

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