PMID: 2497771Feb 21, 1989Paper

Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli

Biochemistry
S ClearyR F Kelley

Abstract

We have expressed the 174-263 fragment (kringle-2 domain) of human tissue-type plasminogen activator (t-PA) in Escherichia coli by secretion into the periplasmic space using the alkaline phosphatase promoter and stII enterotoxin signal sequence. A large portion of the secreted protein is associated with an insoluble cellular fraction. This material can be solubilized by extraction with denaturant and reducing agent and then recovered in active form by refolding in the presence of reduced and oxidized glutathione. Kringle-2 is then easily purified by affinity chromatography on lysine-Sepharose followed by cation-exchange chromatography. The isolated protein has an amino acid composition and N-terminal sequence as expected for the 174-263 fragment of t-PA, indicating that the signal peptide has been properly removed. Circular dichroic spectra suggest that the protein is folded similar to the kringle-4 domain of plasminogen [Castellino et al. (1986) Arch. Biochem. Biophys. 247, 312-320]. Equilibrium dialysis experiments indicate a single binding site on kringle-2 for L-lysine having a KD of 100 microM. Using a method based on elution of kringle from lysine-Separose with omega-aminocarboxylic acids [Winn et al. (1980) Eur. J. Bioch...Continue Reading

References

Dec 1, 1977·Proceedings of the National Academy of Sciences of the United States of America·F SangerA R Coulson
Nov 24, 1979·Nucleic Acids Research·H C Birnboim, J Doly
Jun 1, 1986·Archives of Biochemistry and Biophysics·F J CastellinoJ M Beals
Jul 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·A J van ZonneveldH Pannekoek
May 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·M Trexler, L Patthy
Oct 25, 1984·Journal of Molecular Biology·V V NovokhatnyP L Privalov
Sep 1, 1982·Hoppe-Seyler's Zeitschrift für physiologische Chemie·G J SteffensL Flohé
Mar 3, 1981·Biochemistry·J P Hennessey, W C Johnson
May 29, 1981·Biochimica Et Biophysica Acta·S ThorsenS Magnusson

❮ Previous
Next ❯

Citations

Jul 17, 1999·The Journal of Peptide Research : Official Journal of the American Peptide Society·Y ChangF J Castellino
May 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·S H BassJ A Wells
May 26, 1998·Biochemical and Biophysical Research Communications·R MoreauP Manjunath
Aug 26, 2010·Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism·George W J HarstonAlastair M Buchan
Apr 1, 1992·Archives of Biochemistry and Biophysics·V S DeSerranoF J Castellino
Aug 9, 2016·Protein Expression and Purification·Mohammad Naser TaheriSedigheh Sharifzadeh
Feb 12, 2005·Preparative Biochemistry & Biotechnology·Hong-Xia ZhangZi-Chun Hua
Nov 11, 2003·Preparative Biochemistry & Biotechnology·Ming ZhuZi-Chun Hua
Nov 19, 1991·Biochemistry·M R RejanteM Llinás
Oct 16, 1990·Biochemistry·M G ObukowiczD T McPherson
Apr 23, 2008·Current Protocols in Protein Science·Paul T Wingfield
Apr 2, 2015·Current Protocols in Protein Science·Paul T Wingfield

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.