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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.


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.


PubMed | University of Sichuan, Sichuan University and Genhunter Corporation
Type: | Journal: Protein expression and purification | Year: 2015

The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.


PubMed | University of Sichuan, Sichuan University and Genhunter Corporation
Type: Journal Article | Journal: 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.


Genhunter Corporation | Entity website

DD still leads in publications! Because of several major advantages, Differential Display (DD) technology continues to lead over other competing technologies such as DNA microarrays and SAGE in citation index and Medline hits (see figure and chart at right). The advantages of DD include: 1) Simplicity: Technically, it is based on PCR and DNA sequencing gel electrophoresis ...


Genhunter Corporation | Entity website

GenHunter has collected a few testimonials over the years. None of these were sought-after or rewarded in any way ...


Genhunter Corporation | Entity website

PerfectWestern Containers 37 different sizes now available Due to the popularity of these containers and based on continuing feedback from our customers, GenHunter has expanded this product line again and now offers 37 different sized containers to fit your needs. For the most up-to-date information, we highly recommend downloading the PerfectWestern brochure by clicking here to obtain all the information including dimensions, catalog numbers, and current pricing Newest products are: Medium size is available in a more flexible "Dura" material and 3-Sectional-Medium option 6-Sectional containers (-Short, -Mid, and -Long) are available in opaque black material New compartmented/sectional sizes: 5-Sectional-Shorter, 6-Sectional-Mid-LS, 6-Sectional-ExtraLong Mini Square: 8 ...


Genhunter Corporation | Entity website

DNA Microarray: Publications Revealing Problems with the Technology Sydney Brenner, Peng Liang, Stephen Cooper, Nat Goodman and others weigh in This is a list of publications that GenHunter has compiled from well-known scientists including recent Nobel Prize winner Sydner Brenner, Richard Klausner, Thomas Cebula, Arthur Pardee, Peng Liang, Stephen Cooper, Nat Goodman and many others. These publications reveal some of the problems and common pitfalls of Microarray technology ...


Genhunter Corporation | Entity website

Would you like to save even more money on Western blots? Use our PerfectWestern Containers with the Best Rocker on the Market: The PerfectRocker! The PerfectRocker offers the standard back-and-forth rocking motion preferred by many labs. But it also includes adjustable speed (2 to 30 RPM) and adjustable pitch (0 to 30 degree tilt angle) ...


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.

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