A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli.
Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.
Kareem
B N
BN
Biotechnology Center of Oslo, Norway.
Rokkones
E
E
Høgset
A
A
Holmgren
E
E
Gautvik
K M
KM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D010281:Parathyroid Hormone; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins