TNF-alpha, a prototype member of the TNF family of ligands, has both pro-inflammatory and immune regulatory functions, which make it as an appropriate therapeutic target for selective blockade in antibody therapy of many diseases like in rheumatoid arthritis. Using two models of SHuffle (R) T7 Express and BL21 (DE3) cells, we have expressed this protein and recognized it by SDS-PAGE analysis. FTIR biospectroscopy of the resulted purified proteins has been performed and mathematical calculations has been done for further identification of the structural and conformational differences between the two products. Our results showed some differences in disulfide bond formation and beta-sheet turns between these two recombinant proteins. To the best of our knowledge, this is the first study that compare secondary structure of recombinant proteins in both conventional and next generation Escherichia coil based expression systems using reliable, simple, rapid and economic ATR-FTIR analysis. Whether these differences might have significant effects on TNF-alpha inflammatory and immune-regulatory function in biological systems might be very much important and need further investigations. (C) 2017 Elsevier B.V. All rights reserved.