By David R. Higgins, James Cregg
David Higgins and James Cregg bring together for the 1st time an all-inclusive selection of key experimental strategies to govern Pichia pastoris for gene expression reasons. The methods-developed via world-class researchers of their personal laboratories-contain designated descriptions of accessible expression traces, in addition to vectors and step by step directions for the development of expression traces and for his or her use within the creation of overseas proteins from shake-flask or fermentor cultures. common tactics for the purification of international proteins also are supplied, besides protocols for studying overseas protein items for glycosylation and subcellular position. Its targeted directions will allow any researcher in molecular biology to speedy build an expression pressure that would produce a wanted overseas protein, and to exploit this cutting-edge molecular and mobile organic procedure effectively in each sector of organic examine at the present time.
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S In such instances, the assay of routine samples is halted until possible causes are checked and corrected. 3). 8 E. Mullins, Analyst 119, 369 (1994). [6] D e t e r m i n a t i o n o f T e t r a h y d r o b i o p t e r i n Biosynthetic Activities by High-Performance Liquid Chromatography with Fluorescence Detection By ERNST R. WERNER, HELMUT WACHTER, and GABRIELE WERNER-FELMAYER Introduction Tetrahydrobiopterin [6R-6(L-erythro-l,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin] serves as cofactor of phenylalanine 4-monooxygenase, tyrosine 3-monooxygenase, glyceryl-ether monooxygenase, and nitric oxide synthase.
Werner-Fehnayer, E. R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter, J. Exp. Med. 172, 1599 (1990). METHODS IN ENZYMOLOGY, VOL. 281 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25 54 v o u c ACID [61 guanosine 5"triphosphate (GTP) GTP cyclohydrolase I I ""~ (no cofactors required) 1 0 OH OH /N~ HN ,~ I II HN " ~ % 2 HCOOH ~L- ~'~l~ I "J~N ~H H H CH20-P30;- 7'8-dihydr°neopterin triph°sphate 6-pyruvoyltetrahydro- [ pterin synthase (requires Mg2÷) o H ~N~ HN 2 o 0 H'~I + N H H CH3 6-pyruvoyltetrahydropterin H sepiapterin reductase 1 (requires NADPH) o HN 3 HN A I L N 8 2 H H 5 6 OH OH 67~OHH tl~:rahydro.
261, 15416 (1986). [41 Microbiological Assay of Folates i n 96-Well Microtiter Plates By DONALD W. HORNE Our understanding of the dietary requirement for, and the metabolism of, the vitamin folic acid rests, mainly, on our ability to determine the amount present in serum, erythrocytes, and other tissues. Several procedures are currently in use for estimating folate concentrations; these include microbiological, radiochemical, and electrochemical assays. These proce- METHODS IN ENZYMOLOGY,VOL. 281 Copyright © 1997by AcademicPress All rightsof reproductionin any form reserved.