Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence.
A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015247:Deoxyribonuclease HindIII; D004926:Escherichia coli / Q000378:metabolism*; D005786:Gene Expression Regulation; D006629:Hirudins / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology
D006629:Hirudins; D021382:Protein Sorting Signals; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII; D000469:Alkaline Phosphatase; D013917:Thrombin