Tetrameric hemoglobin expressed in Escherichia coli. Evidence of heterogeneous subunit assembly.

Recombinant alpha 2 beta 2 tetrameric Hb expressed and assembled in Escherichia coli has been characterized extensively. Electrospray mass spectrometry and optical and electron paramagnetic resonance spectroscopy suggest that the overexpressed protein is identical to native human Hb. Although the functional properties of this recombinant Hb are nearly identical to native Hb, crucial differences exist between the two molecules. The recombinant Hb expressed in E. coli has a lower Hill coefficient even though oxygen equilibrium binding studies indicate cooperative binding. The most significant difference observed between the recombinant and native Hb is the loss of oxygen affinity regulation by 2,3-diphosphoglyerate and protons. CO binding to the deoxy tetramer was found to be biphasic with both phases sensitive to the presence of allosteric effectors. The recombinant chains were isolated, and the ligand binding properties demonstrated that the recombinant chains behave in a similar fashion to native alpha-sh and beta-sh. To investigate whether the chains were capable of forming a well behaved tetramer, the isolated chains were reassembled into a tetramer and purified to homogeneity. Oxygen binding properties of the reassembled recombinant Hb now show an increased Hill coefficient of 2.5, close to, but still slightly lower than, that observed for native Hb. Additionally, reassembly of recombinant Hb produces a protein that is subject to regulation by allosteric effectors. Furthermore, CO binding to the reassembled recombinant deoxy tetramer was found to be monophasic under all conditions.

Human hemoglobin expression in Escherichia coli: importance of optimal codon usage.

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. alpha-Globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains. N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue. Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.