Expression of recombinant monomer hemoglobins (component IV) from the marine annelid Glycera dibranchiata: evidence for primary sequence positional regulation of heme rotational disorder.
A description of the efficient high-level expression of the monomer hemoglobin (GMG4) from Glycera dibranchiata is presented. The cDNA described by Simons and Satterlee [Simons, P.C., & Satterlee, J.D. (1989) Biochemistry 28, 8525-8530] was subcloned into an expression system, and conditions were found that led to the production of large amounts of soluble apoprotein (rec-gmg). These conditions included lowering the temperature during the induction period and growth in a rich medium with a higher ionic strength. Characterization of this reconstituted recombinant protein showed that it was not identical to the native GMH4 protein. Both UV-visible and 1H NMR data indicated differences within the holoprotein (rec-gmh) heme pocket compared to the native protein, the major difference being that two nonidentical heme orientations are significantly populated in rec-gmh. This phenomenon has been seen previously in other heme proteins, where these heme orientational isomers are described by a 180-deg rotation about the heme alpha-gamma meso axis. This work prompted the production of a complete chemical sequence for the native GMH4 [Alam S.L., Satterlee, J. D., & Edmonds, C. G. (1994) J. Protein Chem. 13, 151-164], which showed that the expressed rec-gmg protein differed at three primary sequence positions (41, 95, and 123) from the native component IV globin (GMG4). Subsequently, we have produced the triple-revertant mutations required to express the recombinant wild-type protein (recGMG4). The physical characteristics of the active site in the holoprotein (recGMH4) are identical to those of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Alam
S L
SL
Department of Biochemistry, Washington State University, Pullman 99164.
Dutton
D P
DP
Satterlee
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D000845:Annelida / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013053:Spectrophotometry
D018076:DNA, Complementary; D006454:Hemoglobins; D011994:Recombinant Proteins; D006418:Heme