We have separately expressed the catalytic domain (R3) of beta-1,4 galactosyltransferase (beta-1,4 GT) and the full length enzyme, containing the transmembrane domain, in E.coli. The recombinant proteins are present in inclusion bodies as insoluble precipitates which do not exhibit any enzymatic activity. Previously a method was developed in this laboratory to generate soluble and active protein from the inclusion bodies. This method has been modified and improved to increase the solubility of the protein that is required for the crystallization purposes. Using this methods we are now able to produce enough quantity of R3 protein for crystallization studies. Preliminary crystallization studies indicate that the R3 protein crystals can be obtained in a period of 1 to 3 days. Attempts are being made to grow better quality and suitable size crystals for x-ray crystallographic investigations. To further improve the solubility of the recombinant protein, Phe residues, which might contribute to the low solubility were mutated. By site directed mutagenesis all Phe residues were mutated to Ala, one residue at a time and the mutated protein tested for the solubility. The results indicate that replacing the Phe residue at 149 or 226 or 236 with Ala, the solubility increases by three fold. However, when Phe at 149 is replaced by Lys there was no change in solubility. Further detailed investigations are being carried out. Similar to beta-1,4 GT, large amount of recombinant mouse alpha-lactalbumin, a modifier protein of beta-1,4 GT, is obtained as inclusion body in E.coli. We are able to produce large quantities of active and soluble protein from the inclusion bodies, using the method developed for beta-1,4 GT with a slight modification. Preliminary crystallization studies indicate that this protein crystallizes at room temperature using the conditions quite similar to the one used for human alpha-lactalbumin.
