Expression and Purification of apo-Micro-Myoglobin and its Reconstitution to the Heme binding Micro-Myoglobin
M. Haeubl1, M. Kriegl1, R. Goessler1, W. Schoefberger1, N. Müller1
of Organic Chemistry, Johannes Kepler University Linz,
Altenberger Str. 69 4040 Linz Austria
Apo-µ-Myoglobin is a heme binding 78-amino-acids fragment of sperm whale myoglobin. The unlabeled and 15N-labled apo-µ-myoglobin were expressed in E. coli BL21(l DE3)-cells containing the plasmid pSSB1 [1, 2]. One of the biggest problems after the protein expression was precipitation of this protein. During our investigation we found proper conditions where apo-µ-myoglobin shows its highest solubility.
We used CD-measurements, UV-vis spectra, COSY, TOCSY and NOESY NMR measurements for the unlabeled apo-µ-myoglobin. While the 15N labeled compound was investigated by preliminary 15N-HSQC NMR to verify the proteins folding conditions.
In the next step we prepared unlabeled holo-µ-myoglobin. This was done in a reconstitution process where the apo-µ-myoglobin was mixed with an equimolar amount of heme which binds to µ-myoglobin and changes the proteins folding conditions. Due to this change the surface of µ-myoglobin gets more hydrophobic and the protein is more likely to precipitate. We found suitable conditions for the reconstitution of apo-µ-myoglobin and CD-measurements of the holo-µ-myoglobin suggested a change in the folding state of holo-µ-myoglobin. This change of folding is also visible in the UV-vis spectra where the absorption maximum shifts from 275 nm to 412 nm.  The heme itself absorbs at 385 nm. Unfortunately the holo-µ-myoglobin is very likely to precipitate in the chosen buffer. Therefore any attempts to concentrate the reconstituted protein for NMR measurements failed. To keep the protein soluble we changed the buffer conditions and the proteins solubility increased significantly. This route seemed to be a very promising way to achieve the necessary concentrations for NMR experiments.
 Grandori R, Schwarzinger S, Müller N.; Eur.
J. Biochem. 2000; 267: 1168-1172.
 Schwarzinger S, Ahrer W, Müller N.; Chemical Monthly 2000; 131: 409-416.