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Download e-book for kindle: Applications of Chimeric Genes and Hybrid Proteins Part A: by Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N.

By Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N. Abelson

ISBN-10: 0121822273

ISBN-13: 9780121822279

The significantly acclaimed laboratory common for greater than 40 years, Methods in Enzymology is without doubt one of the such a lot hugely revered guides within the box of biochemistry. due to the fact 1955, each one quantity has been eagerly awaited, often consulted, and praised by way of researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence comprises a lot fabric nonetheless appropriate today--truly a vital booklet for researchers in all fields of existence sciences

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Extra resources for Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification

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Beckwith, J. Mol. Biol. 130, 303 (1979). 26 j. D. Hopkins, Z Mol. Biol. 87, 715 (1974). 34 GENE FUSIONS [2] given fusion strain. Because a relatively high level of LacY expression is required, it may not be possible to apply TONPG selection to fusion strains that already have low activity. Selecting for Increased Expression of Reporter Fusions A different galactoside, phenylethyl-/3-o-thiogalactoside (TPEG), is a competitive inhibitor of/3-galactosidase, 27 and can be used to select for mutations that increase the expression of lac operon fusions with low levels of expression.

All cells, which can grow on lactose tetrazolium agar, reduce the tetrazolium (2,3,5triphenyl-2-N-tetrazolium chloride) to form a red, insoluble formizan dye. 24 j. p. Horwitz, J. Chua, R. J. Curby, A. J. Tomson, M. A. DaRooge, B. E. Fisher, J. Mauricio, and I. Klundt, J. Med. Chem. 7, 574 (1964). 32 GENEFUSIONS [21 The acid by-products secreted by Lac ÷ colonies inhibit the reduction, preventing formation of the formizan. Therefore, Lac ÷ colonies are white on tetrazolium, whereas Lac- colonies are red.

Different schools of thought exist on the choice of phage and plasmids for the integration of fusions onto the chromosome. Some researchers 13j. M. Robertson and J. Walsh-Weller,Methods Mol. Biol. 98, 121 (1998). 18 GENE FUSIONS [21 prefer to use combinations that produce lysogens in which the fusion is linked to a drug resistance marker. This facilitates the movement of the fusions from one strain to another by generalized transduction. Paradoxically, marked fusions can complicate strain construction because of the loss of the ability to use the marker on the A phage to move other mutations or maintain plasmids.

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Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification by Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N. Abelson

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