Effects of amino acid insertions on the proteolysis of a staphylococcal protein A derivative in Escherichia coli.
In vivo proteolysis of protein ZZT0, derived from the B domain of staphylococcal protein A, was investigated in Escherichia coli before and after insertion of 1-3 multiples of the tetrapeptide Ala-Trp-Trp-Pro close to the C-terminus of ZZT0. Before insertion, ZZT0 was proteolytically stable as judged from the purity of IgG binding proteins up to 1 h after inhibition of protein synthesis with chloramphenicol. Insertion of 1-3 units of Ala-Trp-Trp-Pro into ZZT0 increased progressively the sensitivity to proteolysis and induced DnaK and GroEL binding to the protein. The time for 50% in vivo hydrolysis of the full length protein derivative that was most susceptible to proteolysis, i.e. with three tetrapeptide units, was about 40 min when cultivated in a bioreactor and about 4 min in a shaken flask culture. Molecular masses and N-terminal sequences of the main degradation products indicated that protein ZZT0 is cleaved at identical sites irrespective of the number of inserted tetrapeptide units and that the cleavage sites are located far from the insertion point. Insertion of another hydrophobic amino acid, isoleucine, as the tetrapeptide Ala-Ile-Ile-Pro, only induced a slight proteolysis of the ZZT0 molecule under similar conditions. This indicates that the insertion of tryptophan residues, rather than of a general hydrophobic segment, plays an essential role in the induced proteolysis of the ZZT0 protein.
Yang
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S
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Bergman
T
T
Veide
A
A
Enfors
S O
SO
D000595:Amino Acid Sequence; D018834:Chaperonin 60 / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D010450:Endopeptidases; C063842:dnaK protein, E coli