Abstract: The expression level of penicillin G acylase (PAC) gene in different host strains harbouring same plasmid (pKK SP) were different, among them, the highest expression was achieved in E.coli BL21(DE3), the lowest in E.coli BL21 and moderate expression levels were observed in E.coli D816 and E.coli HB101. The special activity of penicillin G acylase gene in four host strains are all dependent on molecular chaperon GroEL content in these cells. Analysis of results from sodim dodecyl sulfate/polyacrylamide gel electrophoresis and immuno blot of whole cell proteins in these cells indicated that folding of subunits were bottleneck steps limiting posttranslational process of penicillin G acylase. The posttranslational efficiency (folding rate, secretion rate) of PAC is dependent on molecular chaperon GroEL content in these cells.