Peptidoglycan (PG) hydrolases or autolysins are a group of enzymes which degrades bacterial cell wall at specific sites. Staphylococcus aureus produces two major PG hydrolases: major autolysin (AtlA) and Aaa, a autolysin/adhesin protein. Amidase are surface-associated proteins that have both enzymatic and adhesive functions. AtlA is the most predominant autolysin in Staphylococcus aureus. The AltA protein only with an amidase domain and the two repeat sequences R1 and R2 is 62 kDa. Another autolysin/ adhesin present on the cell surface of S. aureus (Aaa) is a 35 kDa protein containing two direct LysM (lysine motif) repeats at the N-terminal and catalytic domain in the C-terminus. The amidase domain of the atlA gene with two repeat regions (amiE-R1,2) was cloned and expressed as N-terminal His-tag fusion protein in pQE30 (Qiagen) vector in E.coli M15 (Biswas et al., 2006). For the over-expression of His-tag Aaa; the aaa open reading frame was cloned in the pET28 (Novagen) vector designed to express proteins as fusions with a His6-tag at the N-terminus (Biswas R, 2006). We grew the E. coli M15 cells carrying the altA gene encoding the His-tagged protein in LB medium containing ampicillin to an OD600 nm of 0.5; expression was then induced with different concentrations of isopropyl β-D-thiogalactoside (IPTG). Cells were harvested by centrifugation, and lysed by freeze thaw method with the addition of lysozyme. Proteins were purified under denaturing conditions with 8M urea and analysed by 10 % SDS PAGE. In the same way, we also cultured the E. coli BL21 cells carrying the aaa gene but in LB medium with kanamycin instead of ampicillin. The optimal IPTG concentration for both was found to be 1mM. Isolation of insoluble and soluble fractions of recombinant protein from the bacterial cell lysate and their analysis confirmed the presence of both the recombinant proteins in the insoluble fraction possibly as inclusion bodies. Large scale purification of both the recombinant proteins was done using nickel (Ni-NTA) affinity chromatography. Maximum protein elution was found in 250mM imidazole buffer in both the cases. Refolding was optimized in refolding buffer containing 0.5mM reduced glutathione: 0.5mM oxidised glutathione (1:1) in 50mM TrisHCl pH-8.0. After refolding the bioactivity of both the proteins were assayed. Cell lysis activity of the refolded protein was observed to be more for 61KDa protein than the 35KDa protein. The refolded 61kDa amidase was found to be strongly binding to fibronectin found by affinity column chromatography.