Expression of a plant codon optimized pat gene encoding phosphinothricin acetyltransferase (PAT) in bacterial expression systems required modification of the 5' end of the pat ORF. Modifications necessary for improving the expression were identified by a coupled in vitro transcription and translation process. The dramatic improvement in the expression of PAT was due to the removal of a potential secondary structure that could have resulted in the inhibition of translational initiation. Therefore, in vitro transcription and translation is a versatile tool to optimize gene sequence for protein overexpression. Additionally, this method was shown to be successful in both Escherichia coli and Pseudomonas fluorescens. Gene sequence optimization and choice of host along with cultivation conditions also had major impact on PAT expression. P. fluorescens was a better host than E. coli resulting in 30-fold more expression of PAT. We were able to recover approximately 95mg of purified PAT from P. fluorescens using a three step chromatographic process.