Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli.
A 1,820 bp full-length clone encoding for a new human protein was isolated from a lambda gt11 placental cDNA library using anti-human hexokinase antibodies. The cDNA complete sequence includes a 12 bp 5' non-coding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 bp non-coding with two putative polyadenylation signals upstream of 3' poly(A)tail. The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274. Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences. In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein. Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins. Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E. coli.
Daniele
A
A
Dipartimento di Genetica, Biologia e Chimica Medica, Università di Torino, Italy.
Altruda
F
F
Ferrone
M
M
Silengo
L
L
Chiarantini
L
L
Bianchi
M
M
Stocchi
V
V
Magnani
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006593:Hexokinase / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010920:Placenta / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D001426:Bacterial Proteins; D011506:Proteins; C073187:HP-10 protein, human; D006593:Hexokinase