The plasmid pMSP3535 is a popular vector for nisin-inducible expression of heterologous genes in lactic acid bacteria. However, the maximum protein expression level achievable with pMSP3535 is relatively low. In an effort to increase expression we modified pMSP3535 to create a high expression variant termed pMSP3535H2. Modifications included removal of a small NisA peptide fragment from the P nisA promoter and addition of a bidirectional transcription terminator. In addition the plasmid copy number was increased by replacing the pMSP3535 copy number control region with that of a high copy variant of the same replicon. As a result of these modifications, expression of two target proteins, the green fluorescent protein and the Escherichia coli antigen intimin, increased 5.0- and 7.5-fold, respectively. The increased range of inducible expression achieved with pMSP3535H2 will facilitate molecular studies in a range of lactic acid bacteria.