The majority of current high-throughput protein purification protocols include rate-limiting centrifugation steps. A column and nozzle assembly was developed that can be used in-line with microfluidization for the purification of bacterially-overexpressed, His-tagged proteins directly from bacterial cultures. Yields and purity are comparable with standard protocols. This large-scale protein purification protocol is easy to use and widely-applicable. (C) 2011 Elsevier Inc. All rights reserved.