Kai, Lei (2012)
Development and application of a high-throughput cell-free expression system.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

At the post genomics age, the study of proteomics attracts more and more attention and help to decipher the hidden information inside genes. However, the study of proteomics required large amount or large number of proteins to be synthesized. During the past several decades, heterologous overexpression of recombinant protein from bacteria, yeast, insect cells and mammalian cells was developed. Especially the E. coli system, which is the most studied expression system, was used to obtain different recombinant proteins from both prokaryotic and eukaryotic organisms. Still, there are a large number of eukaryotic proteins that cannot be functionally overexpressed in the E. coli system. Techniques were developed to overexpress recombinant protein in eukaryotic host cells including yeast, insect cells and mammalian cells. However, these eukaryotic overexpression systems normally require more complicated processes and take more time to finally get the protein. Cell-free (CF) expression systems, using cell lysates, which contain the required enzymes, extra energy resources and substrates needed for translation, were developed. The CF system was first used to discover the genetic code by in vitro translation of poly-U RNA sequences. Later on, it was applied to protein synthesis, attracted more attention and became an alternative method for protein expression besides traditional cell based expression systems. Eliminating the need of viable cells opens the CF expression system for various modifications. The CF expression system was widely used to express difficult target proteins like toxins, membrane proteins (MPs), insoluble or aggregation-prone proteins that failed to be expressed in in vivo. The open nature and emendation for throughput automation made the CF system an ideal system for proteome research. In this thesis, a high throughput CF expression platform, in particular, addressing difficult targets including MPs and aggregation prone proteins is developed. In order to establish the high-throughput CF expression platform, a batch configuration which is less time consuming, was selected. The amount of protein expressed in low-volume batch configurations can be addressed by more sensitive detection methods. With the TECAN Freedom EVO 200 liquid handling robot, the full automation of the CF expression system was achieved. This platform provides parallel expressions in 96-well plates with a minimum volume of 25 µl. The whole expression takes 2-4 hours with an expression yield above 0.5 mg per 1 ml reaction mixture. A custom designed software E.Y.E.S which provides a more versatile and friendly user interface to design the screening of CF expression conditions, was created. With the help of E.Y.E.S program the time for robot programming and pipetting was largely reduced. The whole pipetting time was reduced to 30-45 minutes for 96 reactions. The high-throughput expression optimization can be combined with fluorescence detection by fusing shifted green fluorescent protein (sGFP) to the target protein, allowing a fast read out. With the established high-throughput CF expression system, we systematically screened the effect of common chemical stabilizers. The sGFP was chosen as a target protein. In the first step, the compatibility of the selected chemical stabilizers was evaluated by measuring the fluorescence of CF expressed sGFP. With these experiments, the effect of selected stabilizers can be classified into three different types: positive, negative and tolerant. In case of a positive effect, the selected stabilizer increased the expression or folding of sGFP, which resulted in an increase of the measured fluorescence. A negative effect indicated that selected chemical compounds were toxic to the CF expression system and inhibited the expression at low compound concentrations. A tolerant effect represented that the expression of sGFP in CF system had a linear decrease with the increase concentration of this type of chemical stabilizers. Those stabilizers which had a positive or tolerant effect on the CF expression system can be used and applied to those unstable protein or aggregation prone proteins to improve the solubility and stability of the sample as soon as they were expressed. In order to check the application of these chemical stabilizers, we choose two targets. One is human GNA1 (EC 2.3.1.4) which catalyzes the transfer of the Acetyl group from Acetyl Coenzyme A (AcCoA) to the primary amine of D-glucosamine-6-phosphate to form N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6P) and Coenzyme A (CoA). Another target is CurA-halogenase, which is the first enzyme within the biosynthesis of curacin A (a bioactive compound with potent anti-proliferative activity) pathway. The biosynthesis of CurA is mediated by a 2.2 MDa hybrid polyketide synthase (PKSs) and non-ribosomal peptide synthetases (NRPSs). Both of the two targets were expressed as partially soluble in vivo and in vitro. We selected several chemical stabilizers to check the ability of these chemical stabilizers to improve the soluble expression of the selected two targets in CF expression system. For the case of halogenase, a maximum of around 25% increased soluble expression was observed with choline. For GNA1, a beneficial effect of choline was also observed. To further check if the fluorescence signals from the protein fused with sGFP correlated with the function of the fusion partners, we created a GNA1-sGFP construct which sGFP was fused at the C-terminal of GNA1. Results of the experiment showed that the function of GNA1 was not always correlated with the fluorescence signal from sGFP. In the case of L-arginine, the fluorescence of the fusion protein was increased without the increase of the functionality of GNA1, which might explained by that L-arginine had a beneficial effect on the sGFP folding. While for the case of choline, the increase of fluorescence signal correlated with the functional assay. Finally, in order to provide an example of correlated screening of two chemical compounds, we select PEG8000 and choline to perform this experiment. When using PEG8000 alone, an increase of soluble expression was observed around 17% was observed. While with choline alone around 23% more soluble expression was achieved. While when using both PEG8000 and choline at the optima concentrations, an increase of around 60% was obtained. This 60 % increase was much more than simply sum up of the 17% and 23%. Which might indicate an additional beneficial effect came from the interaction of the two compounds. The successful application of the systematically screening of chemical stabilizers offered an example to use the automatic throughput CF platform. Preliminary studies on the application of the established throughput CF platform on MPs were performed. We choose the D-CF mode for directly soluble expression of MPs in this platform. However, one challenge was to find a monitor to characterize the expression and even the folding of expressed MPs. Taken the idea from in vivo expression system for MPs, which use a C-terminal GFP fusion for the fast detection of soluble expressed MPs in either E. coli or yeast expression system. The same approach was used in this CF system. By following the fluorescence of the fused sGFP, the soluble expression of MPs can be detected and quantified. Since detergents was used to provide hydrophobic environment for MPs, the folding of GFP in presence of detergents needs to be evaluated. However, results showed that the folding of normal sGFP was highly influenced by detergents. sGFP only gave relative high fluorescence accounts in limited detergents like Brij derivatives. With the best detergent, less than 60% sGFP was folded compared to the control without detergent. With other commonly used detergents like Digitonin, TritonX-100 and DDM very low fluorescence signals were detected, indicating that sGFP was poorly folded in these detergents. The limitation of choosing detergents reduced the application this method for many MPs because many MPs cannot be soluble expressed with the Brij detergents. In order to solve this problem, a mutant of GFP was selected, which was called superfolderGFP. This superfolderGFP was super stable, less affected by the out environment and fast folding compared to normal sGFP. A comparison experiment of detergent screening was performed with both sGFP and superfolderGFP showed that in general the superfolderGFP had a better detergent tolerance. Those detergents like TritonX-100, Digitonin and DDM can allow the folding of superfolderGFP to more than 70%. With Brij-78 the fluorescence was almost the same compared to the control. Furthermore, a MP SugE (small multidrug transporter) was selected as an example for detergent screening. Result showed that SugE fused with superfolderGFP gave relative good fluorescence signal in Brij-78, Brij-58, Digitonin and Lauryl-MNG. With Brij-58, the fluorescence was even higher than Brij-78. These two experiments showed that with superfolderGFP more detergents can be select for D-CF expression of MPs fusion partners. Introducing the superfolderGFP as fusion monitor increases the application of the throughput CF platform in detergents screening for MPs. Further experiment with detergents and lipids mixture provide another promising tool for obtain soluble MPs with functions. However, more research still need to be done to provide more information about the ratio of lipid/detergents in practical applications. Two different MPs, SugE (small multidrug transporter from E. coli), AQP4 M23 (aquaporin 4 from mouse), were selected and studied. The sample preparation of SugE, offered a new approach for obtain pure, homogeneous and stable MP protein sample. SugE was expressed in CF system first as precipitate and later on re-solubilized with detergent. A two-step re-solubilization strategy was applied. First treat the protein pellet from the reaction with relative mild detergent like NDSB256. Second, re-solubilize the remaining pellet with DDM. The first step, the mild detergent will solubilize quite a lot of impurities not the target protein SugE. In the second step, DDM will solubilize most of the SugE protein and small amount of impurities. With this two-step re-solubilization approach, without any chromatography approach, the sample was already relative pure. Later on with another SEC, the sample was purified as one pure band on SDS-PAGE and showed homogeneous elution profile on the gel filtration column. Later on, in order to obtain more detergent conditions which can keep SugE homogeneous and stable, a detergent exchange was performed directly on the SEC column. Detergents with different micelle size were selected and some of them also showed comparable elution profile of DDM. Another MP we selected was AQP4 M23. The mouse aquaporin 4 was selected as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based CF system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (CF MP expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (CF MP expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of CF reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated Pf value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10oC, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.

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