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Recombinant porcine myoglobin has been produced in Escherichia coli using the lambda cII fusion expression system of Nagai and Thøgersen [Nature, 309, 810-812 (1984)]. After processing and reconstitution with haem, the protein is gel-electrophoretically and spectrophotometrically indistinguishable from native pig myoglobin. Large crystals of both native and recombinant porcine myoglobin were grown from 50 mM sodium phosphate, pH 7.1, 80% ammonium sulphate. The crystals belong to space group C2 (a = 156.9 A, b = 42.0 A, c = 92.2 A, beta = 127.9 degrees) and diffract to a nominal 2.5 A resolution. We plan to explore apomyoglobin as a binding surface in studies combining site-directed mutagenesis and X-ray analysis. These experiments will be extended by studying the binding of haem analogues to the mutant apoproteins.

We isolated a cDNA clone for myoglobin mRNA from fetal bovine skeletal muscle using a DNA fragment of human myoglobin exon 2 as a probe. The complete coding sequence of myoglobin as well as the 3'- and part of the 5'-nontranslatable sequences (546 and 66 basepairs, respectively) were determined. The amino acid sequence predicted from the nucleotide sequence was in agreement with that determined in the purified protein from adult bovine cardiac muscle (Han, K. K., Dautrevaux, M., Chaila, X., & Biserte, G. [1970] Eur. J. Biochem. 16, 465-471), except for eight amino acid residues: Val-99----Ile,Ile-101----Val, Asn-122----Asp, Ala-124----Gly, Gly-129----Ala, Ala-142----Met, Glu-144----Ala, and Lys-145----Gln. When the myoglobin cDNA was expressed in Saccharomyces cerevisiae under the control of the GAL7 promoter, myoglobin was synthesized as a functionally active holoprotein which bound molecular oxygen reversibly. The amount of myoglobin reached nearly 1% of the total extractable protein in the yeast. N-terminal sequence analysis of the produced myoglobin revealed a glycine residue at the terminus, indicating that as in native muscle the N-terminal Met was removed in yeast by processing.