Purification and characterization of human pancreatic polypeptide expressed in E. coli.
The region of cDNA encoding human pancreatic polypeptide (hPP) was obtained by polymerase chain reaction (PCR) and subcloned into an expression vector. The pancreatic polypeptide gene was expressed in Escherichia coli in two versions: as a cleavable fusion protein with IgG-binding synthetic ZZ domains of protein A from Staphylococcus aureus or with the 1-48 fragment of lambda Cro repressor. Site-specific hydrolysis by hydroxylamine was used to cleave the fusion protein, releasing the human polypeptide. The structure of the obtained hPP has been studied by scanning microcalorimetry and circular dichroism spectrometry. It has been shown that hPP in solutions close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is stable at 20 degrees C and co-operatively breaks down upon heating from this temperature.
Griko
Y V
YV
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Kapanadze
M D
MD
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010191:Pancreatic Polypeptide / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010191:Pancreatic Polypeptide