Cell free protein synthesis versus yeast expression - A comparison using insulin as a model protein.

Expression of recombinant proteins traditionally require a cellular system to transcribe and translate foreign DNA to a desired protein. The process requires special knowledge of the specific cellular metabolism in use and is often time consuming and labour intensive. A cell free expression system provides an opportunity to express recombinant proteins without consideration of the living cell. Instead, a cell free system relies on either a cellular lysate or recombinant proteins to carry out protein synthesis, increasing overall production speed and ease of handling. The one-pot cell free setup is commonly known as an in vitro transcription/translation reaction (IVTT). Here we focused on a PURE (Protein synthesis Using Recombinant Elements) IVTT system based on recombinant proteins from Escherichia coli. We evaluated the cell free system's ability to express functional insulin analogues compared to Saccharomyces cerevisiae, a well-established system for large scale production of recombinant human insulin and insulin analogues. Significantly, it was found that correct insulin expression and folding was governed by the inherent properties of the primary amino acids sequence of insulin, whereas the eukaryotic features of the expression system apparently play a minor role. The IVTT system successfully produced insulin analogues identical in structure and with similar insulin receptor affinity to those produced by yeast. In conclusion we demonstrate that the PURE IVTT system is highly suited for expressing soluble molecules with higher order features and multiple disulphide bridges.

Septin9 is involved in septin filament formation and cellular stability.

Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.