Amino acid content of recombinant proteins influences the metabolic burden response.
Recombinant protein production in Escherichia coli often results in a dramatic cellular stress response best characterized by a decrease in overall cell fitness. We determined that the primary sequence (the amino acid sequence) of the recombinant protein alone plays an important role in mitigating this response. To do so, we created two polypeptides, modeled after the 39-40 amino acid Defensin class of proteins, which contained exclusively the five least (PepAA; His, Trp, Tyr, Phe, Met), or most (PepCO: Ala, Glu, Gln, Asp, Asn) abundant amino acids in E. coli. We determined that overexpression of PepAA resulted in a drastic decrease in growth rate compared to overexpression of PepCO, our model Defensin protein MGD-1, or the 26 amino acid polypeptide contained within the pET-3d vector backbone. We further determined, using Affymetrix E. coli gene chips, that differences among the whole-genome transcriptional responses of these model systems were best characterized by altered expression of genes whose products are involved in translation, transport, or metabolic functions as opposed to stress response genes. Based on these results, we confirmed that translation efficiency was significantly reduced in cells overexpressing PepAA compared with the other model polypeptides evaluated.
Bonomo
Jeanne
J
Department of Chemical and Biological Engineering, Campus Box 424, University of Colorado, Boulder, Colorado 80309, USA.
Gill
Ryan T
RT
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D023082:Defensins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000502:physiology; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013329:Structure-Activity Relationship
D000596:Amino Acids; D023082:Defensins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins