+ Resolve Article
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter

+ Translate
+ Subscribe to Site Feed
GeoScience Most Shared ContentMost Shared Content

High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris

, : High-level secretory production of recombinant bovine enterokinase light chain by Pichia pastoris. Journal of Biotechnology 108(2): 185-192

Enterokinase (EC is a serine proteinase with a specific digest sequence (Asp)4-Lys in the duodenum. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for an in vitro cleavage of fusion proteins. In this work, an active bovine enterokinase light chain (EK(L)) was produced in secretory form by a recombinant strain of the methylotrophic yeast Pichia pastoris. The influences of methanol utilization phenotype of the host strain, induction pH, and carbon source on the recombinant production were studied. The production of recombinant EK(L) by Mut(s) strain was much higher than that by Mut+ strain. When inducted at pH 6.0, on a glycerol/methanol medium, the concentration of recombinant EK(L) (rEK(L)) reached 350 mg l(-1), which was 20-fold higher than that reported previously. The recombinant EK(L) was purified in a simple procedure on the anion exchange chromatography and 15 mg pure active EK(L) were obtained from 100 ml culture broth supernatant. The specific activity of purified rEK(L) was approximately 9000 u mg(-1). To facilitate purification and removal of rEKL after cleavage of fusion protein, the C-terminal His-tagged EK(L) (EK(L)/His) was also expressed in P. pastoris, and this His-tagged EK(L) exhibited a similar enzymatic activity to the untagged EK(L).


PMID: 15129728

DOI: 10.1016/j.jbiotec.2003.11.004

Other references

Elinskii M.P., 1968: Nervous system disorder with decompression disease. Sbornik Trudov Leningradskogo Nauchnogo Obshchestva Nevropatologov i Psikhiatrov (13): 615-622

Wen, R.; Zhang, S.Cheng.; Michaud, D.; Sanfaçon, Hélène., 2004: Inhibitory effects of cystatins on proteolytic activities of the Plum pox potyvirus cysteine proteinases. In an effort to develop new antiviral strategies effective against potyviruses, several cystatins were evaluated for their ability to inhibit the cysteine proteinases of Plum pox potyvirus (PPV) using in vitro proteolytic assays. The following cys...

Labie C., 1986: Current problems in the hygiene of food products listeria monocytogenes and milk products. Revue de Medecine Veterinaire (Toulouse) 137(10): 633-634

Simonova, E.G.; Galkin, V.V.; Loktionova, M.N.; Ladnyĭ, V.I., 2010: Anthrax cattle burial grounds in Russia and their biosafety. To develop standard operating procedure for assessment level of biohazard for population living near existing cattle burial grounds. Development of standard operating procedure for assessment of biosafety of cattle burial grounds was performed on...

Smotherman, W.P., 1984: Learning in the womb. Science 225(4667): 1093-1093

Campbell, G.S., 1977: Thomas G. Orr Memorial Lecture. Surgical physiology of respiratory failure. American Journal of Surgery 134(6): 691-696

Navickas, R.; Petric, V-Kerstin.; Feigl, A.B.; Seychell, M., 2016: Multimorbidity: What do we know? What should we do?. Multimorbidity, which is defined as the co-occurrence of two or more chronic conditions, has moved onto the priority agenda for many health policymakers and healthcare providers. Patients with multimorbidity are high utilizers of healthcare resour...

D.N.poli, S.; Gamba, Z., 2010: Correlated dynamics of water and amphiphilic molecules in thin Newton black films. The dynamical properties of amphiphilics in Newton black films, as well as those of the water confined between the two charged hydrophilic surfaces, have been calculated via a series of molecular dynamic calculations in several films with differen...

Awakura, T., 1994: Cestodes of freshwater fishes of Hokkaido. The cestode parasites of freshwater fishes of Hokkaido, Japan, are listed. The list contains 10 named species and a further 11 forms not identified to species level. The species included are members of the following orders and families: Caryophyll...

Watanabe Y.; Saito A.; Liu H.H.; Hayashi K.; Kawai K., 1991: An epidemiological study on gastric ulcer to duodenal ulcer ration and gastric cancer death in asian countries. The revised gastric ulcer to duodenal ulcer (GU/DU) ratio by hospitals (districts) in Asian countries in 1984 was more than 1 in Kwangju in Korea, Shenyang and Chinghai in China, Manila in the Philippines, and Bangkok in Thailand. And it as less t...