Binding of native bacterial protein SlyD to metal affinity matrices remains a major problem in affinity purification of His-tagged recombinant proteins from Escherichia coli cells. In this study, four novel E. coli strains that lack the expression of SlyD/SlyX, were engineered using lambda-red mediated chromosomal deletion. The resultant mutant E. coli strains allow us to obtain SlyD-free proteins immediately after metal affinity chromatography, and eliminate additional purification processes. As a model protein, bispecific antibodies composed of anti-F4/80 VHH module and anti-TNF VHH module (MYSTI-2) were used. Using this protein we have shown that the SlyD/SlyX-deficient E. coli strains allow us to obtain a fully functional protein. (C) 2018 Elsevier Inc. All rights reserved.