Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions.
Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.
Sharma
Shamik
S
New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USA.
Zhang
Aihua
A
Wang
Haoyong
H
Harcum
Sarah W
SW
Chong
Shaorong
S
D019149:Bioreactors / Q000382:microbiology*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007218:Industrial Microbiology / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing / Q000502:physiology*; D020598:Proto-Oncogene Proteins c-myb / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins