TY - JOUR

T1 - Expression in E. coli systems

AU - Krogsdam, Anne-M

AU - Kristiansen, Karsten

AU - Nøhr, Jane

N1 - Keywords: Escherichia coli; Protein Conformation; Protein Engineering; Recombinant Fusion Proteins

PY - 2003

Y1 - 2003

N2 - Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly (soluble or in inclusion bodies), secreted to the periplasm, or to the surrounding medium. When deciding on a genetic design strategy, it is important to consider the nature of the recombinant protein. The mildest and thus the obvious first-choice expression strategy is to attempt to express the protein intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely to be produced in high yield if secreted from the cell. Secretion eases later purification of the product as the host secretes relatively few of its own proteins. Although tags exist that will direct the protein to the periplasm, only a few reports exist of successfully tagging the protein for extracellular secretion (1). As another strategy to avoid toxicity to the host or if the recombinant protein is very susceptible to cellular proteases, some protection is obtained by targeting the protein to light-refractile aggregates known as inclusion bodies (2,3). Several different strategies exist for subsequent recovery and folding of the protein, which notably must be able to withstand the denaturing-renaturing process.

AB - Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly (soluble or in inclusion bodies), secreted to the periplasm, or to the surrounding medium. When deciding on a genetic design strategy, it is important to consider the nature of the recombinant protein. The mildest and thus the obvious first-choice expression strategy is to attempt to express the protein intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely to be produced in high yield if secreted from the cell. Secretion eases later purification of the product as the host secretes relatively few of its own proteins. Although tags exist that will direct the protein to the periplasm, only a few reports exist of successfully tagging the protein for extracellular secretion (1). As another strategy to avoid toxicity to the host or if the recombinant protein is very susceptible to cellular proteases, some protection is obtained by targeting the protein to light-refractile aggregates known as inclusion bodies (2,3). Several different strategies exist for subsequent recovery and folding of the protein, which notably must be able to withstand the denaturing-renaturing process.

U2 - 10.1385/1-59259-394-1:103

DO - 10.1385/1-59259-394-1:103

M3 - Journal article

C2 - 12840543

VL - 232

SP - 103

EP - 109

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -