title: A revolutionary approach facilitating co-expression of authentic human epidermal growth factor and basic fibroblast growth factor in both cytoplasm and culture medium of Escherichia coli.
abstract: During secretory or excretory production of heterologous proteins in Escherichia coli, peptidase processing cleaves the signal peptide off from a premature protein, which is then secreted as a mature product. Many proteins have been successfully expressed as secreted/excreted products in E. coli. However, basic fibroblast growth factor (bFGF), despite its suitability for secretory/excretory production in E. coli, has never been successfully expressed using such an approach. In this communication, we report the application of a revolutionary E. coli system to the efficient expression of not only bFGF, but also human epidermal growth factor (EGF) concurrently, as authentic products in the culture supernatant (SN). More interestingly, both polypeptides were also shown to be present at high levels as authentic products in the cell lysate (CL). The manifestation of this unusual phenomenon required a collaborative action between construct pWKW2, an efficient excretion vector engineered by our group to facilitate extracellular production of EGF, and the Sce VMA intein, which enables self-cleavage of protein sequences fused to it. Both bFGF and EGF derived from SN and CL were characterized to be bioactive. Moreover, despite employing only shake-flask cultivation, the total yields of bFGF and EGF recovered from both SN and CL were impressive, amounting to 103 and 74 mg l(-1) of culture, respectively. The novel expression approach introduced herein may prove to be practically useful for the production of a wide range of proteins in the future.
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
authors.forename: Keith W Y
authors.initials: KW
authors.lastname: Kwong
authors.forename: W K R
authors.initials: WK
authors.lastname: Wong
mesh_terms: D016475:3T3 Cells; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D003470:Culture Media / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor