Thrombolytic agents are routinely used to dissolve blood clot by activating fibrinolytic system. Among different thrombolytic agents available, staphylokinase (SAK) is gaining much attention because of their fibrin specificity and reduced inhibition by α2 antiplasmin. Though SAK had exhibited less circulatory half life, they are equipotent to tissue plasminogen activator and streptokinase and had shown more potency for clot dissolution during retracted thrombi. In this study, SAK was lipid modified at the N-terminal by a protein engineering approach to enhance its stability and activity. Native SAK as well as the gene encoding SAK with lipobox was cloned into E. coli GJ1158 using pRSET-B expression vector for higher expression. The lipid modification of SAK was confirmed by a mobility shift of 1.3 kDa against the 15.5 kDa of native SAK using tricine SDS-PAGE. Lipid modification of SAK was confirmed by LC MS/MS. The secondary structure analysis was carried out using circular dichroism and deconvoluted fourier transform infrared spectroscopy. LMSAK was found to have a slightly higher denaturation temperature compared to SAK. The improved stablility and activity of lipid modified SAK was studied by heated plasma agar plate assay and mouse tail bleeding test. © 2018