|dc.description.abstract||Recombinant proteins are expressed for use in both research, industry and medicine. The Gram-negative bacteria Escherichia coli (E. coli) is often used as an expression host. It has several properties making it a suitable host, such as fast growth rates and simple protocols for genetic modifications. In addition, it is very well characterized, and has safely been used for a long time as an expression host, and as a laboratory strain. Expressing human or other eukaryotic proteins in E. coli can in many cases be challenging. Expression hosts of human, mammalian, or eukaryotic origin must in some cases be used instead. However, using E. coli or other prokaryotic hosts tend to be cheaper, simpler, and more efficient. As these proteins often are target proteins, especially as pharmaceuticals, overcoming these challenges are of interest.
One strategy for overcoming some of the challenges of using E. coli is translocating the recombinant proteins to periplasm, the space between the outer and inner membrane of Gram-negative bacteria. The environment of the periplasm is different from cytoplasm. This gives some advantages, such as improved folding and solubility. Downward processing is also simplified by periplasmic translocation. In E. coli, proteins can be translocated either post-translationally or co-translationally through the Sec-pathway, or through the Tat-pathway. In this work, components of the Sec-pathway were genetically modified, to increasing the levels of periplasmic translocation for recombinant proteins. The target genes were secM, secG and ffh. The expression of secG and ffh were supposed to be downregulated by changing the ribosomal binding site and the start codon of the genes. The signal peptide of secM were changed to increase the expression of SecA, an important component of the Sec-pathway.
CRMAGE, a combination of λ-Red recombineering based MAGE and CRISPR/Cas9, were used to generate the genomic modifications. Single mutants of secM and secG were succesfully constructed, along with a double mutant harbouring both these mutations. A fusion protein of IgG Fc fragment and sfGFP, with a signal peptide for translocation through the Sec-pathway, were constructed as a reporter. The protein should be in an unfolded state until it has been translocated, thus the fluorescence of sfGFP could serve as a reporter of periplasmic translocation levels. The periplasmic translocation seemed to have been lowered by the introduced mutations. Slightly in the secM mutant strain, and more in the secG mutant strain and the mutant strain harbouring both mutations||