System for the expression of recombinant hemoproteins in Escherichia coli.
Expression of recombinant hemoproteins in Escherichia coli is often limited because a vast majority of the protein produced lacks the heme necessary for function. This is compounded by the fact that standard laboratory strains of E. coli have a limited capacity to withdraw heme from the extracellular environment. We are developing a new tool designed to increase the heme content of our proteins of interest by simply supplementing the expression medium with low concentrations of hemin. This hemoprotein expression (HPEX) system is based on plasmids (pHPEX1-pHPEX3) that encode an outermembrane-bound heme receptor (ChuA) from E. coli O157:H7. This heme receptor, and others like it, confers on the host the ability to more effectively internalize exogenous heme. Transformation of a standard laboratory E. coli protein expression strain (BL-21 [DE3]) with the pHPEX plasmid led to the expression of a new protein with the appropriate molecular weight for ChuA. The receptor was functional as demonstrated by the ability of the transformant to grow on iron-deficient media supplemented with hemin, an ability that the unmodified expression strain lacked. Expression of our proteins of interest, catalase-peroxidases, using this system led to a dramatic and parallel increase in heme content and activity. On a per-heme basis, the spectral and kinetic properties of HPEX-derived catalase-peroxidase were the same as those observed for catalase-peroxidases expressed in standard E. coli-based systems. We suggest that the pHPEX plasmids may be a useful addition to other E. coli expression systems and may help address a broad range of problems in hemoprotein structure and function.
Varnado
Cornelius L
CL
Department of Chemistry, Auburn University, Auburn, AL 36849-5312, USA.
Goodwin
Douglas C
DC
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; C423457:ChuA protein, E coli; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D006418:Heme; D006427:Hemin; D007501:Iron; D010544:Peroxidases; D002374:Catalase