Functional recombinant bovine β-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant β-lactoglobulin such as CD spectra, ligand binding (n, Ka, ΔH, TΔS, ΔG), chemical and thermal stability (ΔGD, Cmid) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine.Electronic supplementary materialThe online version of this article (doi:10.1007/s12033-016-9960-z) contains supplementary material, which is available to authorized users.

Expression of the gene for Batten disease (CLN3) was studied in Escherichia coli and in a cell-free rabbit reticulocyte expression systems. A full-length recombinant fusion CLN3 protein was not produced in the bacterial systems used. However, both N-terminal fragment encompassing 246 amino acids and short C-terminal fragment containing 428-438 amino acids of the CLN3 protein were successfully overexpressed in bacteria. Further studies showed that the C-terminal sequence of the CLN3 protein corresponding to the 356-438 amino acid residues was responsible for inhibition of protein synthesis in bacteria. The full-length CLN3 gene product was readily synthesized in vitro in the cell-free rabbit reticulocyte expression system. The product obtained, corresponding to core CLN3 protein, showed an approximate molecular weight of 43 kDa. Immunoprecipitation of this product with pAb to 4-19 amino acids of the CLN3 protein allows us to suggest that CLN3 protein translation starts at Met-1.