Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45 |
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Authors: | Engels JW; Glauder J; M?llner H; Tripier D; Uhlmann E; Wetekam W |
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Affiliation: | Universit?t Frankfurt, Institut f?r Organische Chemie D-6000 Frankfurt/Main 50
1Universit?t Frankfurt, Institut f?r Organische Chemie Hoechst AG, PO Box 800320, D-6230 Frankfurt/Main 80, FRG |
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Abstract: | By chemoenzymatic synthesis the gene for a (Leu27) analogueof human growth hormone releasing hormone-Gly45 (Leu27GHRH-Gly45]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 1520% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu27)hGHRH-Gly45was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine- -amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference. |
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Keywords: | amidation/ fusion protein/ gene expression in E coli/ GHRH (growth hormone releasing hormone)/ synthetic gene |
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