[Intein-containing chimeric protein construction and evaluation of its cleavage conditions].
Protein splicing is a post-translational autocatalytic process that results in excision of internal peptide (intein) from a precursor protein and the ligation of the flanking protein sequences (exteins). High specificity of the intein-mediated excision of protein precursors allows the use of protein splicing in biotechnology. This work was aimed at the obtaining of human growth hormone with a native N-terminus in E. coli. Chimerical protein consisting of a short N-terminal peptide, Mxe GyrA intein and human growth hormone was created. During the translation formyl-methionine modified N-terminal peptide should have been removed by splicing. Intein was shown to mediate the cleavage of exteins, but their subsequent ligation was not observed. That allowed the preparation of human growth hormone with a native N-terminus. The effect of various factors on cleavage efficiency was studied. The most efficient cleavage of chimeric protein (60-80%) was achieved in the presence of inductor (100 mM beta-mercaptoethanol) upon the incubation for 4-6 days.
Starokadomskiĭ
P L
PL
Dubeĭ
I Ia
IIa
Okunev
O V
OV
Irodov
D M
DM
D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics*; D013006:Growth Hormone / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D047668:Inteins / Q000235:genetics*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D015202:Protein Engineering; D019154:Protein Splicing / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone