Drug development research requires a large amount of target proteins. Screening of drug target often requires 13C- and 15N- labeled protein, and higher protein expression has great advantages for obtaining isotope-labeled proteins. Among many of the proteins, glycosyltransferases(GTs) are attractive biocatalysts in producing a series of important bioactive natural products. In case of kanamycin, kanF gene encodes the first glycosyltransferase which acts both as glucosyltransferase and N-acetyl-glucosaminyl-transferase in kanamycin biosynthetic pathway of Streptomyces kanamyceticus. The recombinant expression of kanF gene in E. coli BL21 (DE3), BL21pLysS and BL21-CodonPlus® (DE3)-RP under T7 promoter-based system showed the expression in insoluble rather than soluble form. Further analysis of the codons revealed that this gene includes number of rare codons which are difficult to be translated in E. coli. Due to variations in codon usage between E. coli and Streptomyces, with high free energy of secondary structure of mRNA, KanF was not expressed under various condition tested. Thus, using codon optimized gene based on codon usage of E. coli has helped to overcome this problem and led to soluble expression of protein. Thus obtained KanF was used for making in vitro reaction for glycosylation of 2-DOS and the product was confirmed to be 2′ -Deamino-2′-hydroxyparomamine by high resolution Q-TOF mass analysis.