Tag Archives: E. coli

The next evolutionary step

Human have always tried to improve on nature, from domestication of plants & animals through directed evolution in the test tube and GMO and up to Craig Venter’s synthetic bacteria and the expansion of the genetic code.
Today, another step was taken towards creating completely artificial life. Continue reading


FISEB 2014 – day 2

Due to crappy Wi-Fi at hotel, this entry will be short. I’ll try to expand once I get back home.

Anyway, today was very interesting.

At the “early bird” session, I heard about CyTOF. Essentially, instead of using a few fluorescent markers for FACS sorting of different cell types, they offer conjugating the tagging antibodies with rare heavy metal isotopes. they claim that these are not found in cells, so the background should be zero. They have >30 different isotopes they can use, and the detection is by mass spectrometry – so very accurate and distinct identification.

Next was a session on gene expression. I won’t go into details, particularly since much is unpublished yet, but Tzachi Pilpel’s talk was amazing. Who knew tRNA may have anything to do with cancer research?

As per usual, Orna Amster-Choder talked about RNA localization in bacteria with lovely images and great data.

Jeff Gerst from Weizmann discovered a possible new mechanism of mRNA transport in yeast, using the MS2 system in very neat ways.

The next session, called “oral poster 1”  featured short talks. The most interesting to me were about mRNA methylation and about how the DNA sequence surrounding consensus sequence for DNA binding proteins affects this binding. some nice insights.

The last session I attended was about the effect of tumor microenvironment on tumor progression and treatments. Heard some amazing stories. Hope still exist to cure cancer…

Tomorrow is my lecture. Excitement!

Those repair crews work fast!

Super-resolution microscopy can potentially allow imaging of single protein molecules. A new paper now tracks single Pol and Lig proteins in E. coli, as they repair DNA damage.

The researchers replaced the endogenous proteins with proteins tagged with a photoactivatable mCherry (PAmCherry). PAmCherry is non-fluorescent, unless activated by UV light (in this case, a 405 nm laser). By using very short pulses, they activate on average less than one molecule per cell at a time. This method is called photoactivated localization microscopy (PALM).

Using this method, they were able to follow single molecules (see this movie), count the number of Pol & Lig proteins per cell, to measure their binding rates to DNA (at optimal and DNA damage states) and importantly, to measure the timing and rates of the different steps of the base excision repair process (BER).

Looking at single repair proteins by PALM. Source: Uphoff S et al. PNAS 2013;110:8063-8068

Looking at single repair proteins by PALM. Source: Uphoff S et al. PNAS 2013;110:8063-8068

Their findings show that Pol & Lig diffuse by Brownian motion near undamaged DNA, but immobilize next to damaged DNA.  Only a small fraction of the proteins (<5%) are involved in repair under normal conditions. But upon severe damage, the excess proteins come into play, to process >1000 DNA damage sites per minute!

The single proteins fix the damage at about 2 seconds per site. That’s pretty fast!

I think that as PALM and other super-resolution methods become more accessible and prevalent, we will be able to collect amazing data on the function of single proteins and other macro-molecules in vivo.

ResearchBlogging.orgUphoff S, Reyes-Lamothe R, Garza de Leon F, Sherratt DJ, & Kapanidis AN (2013). Single-molecule DNA repair in live bacteria. Proceedings of the National Academy of Sciences of the United States of America, 110 (20), 8063-8068 PMID: 23630273

Folding and maturation, or how to evolve your own GFP (part 1)

GFP is one of the most widely used proteins in research. Its usefulness has advanced our understanding of biology in huge leaps forward. One of the greatest advantages of GFP is that the chromophore is formed in an autocatalytyic manner, no need for an enzyme or cofactor assistance. All that is required is atmospheric oxygen.

GFP has sort of a barrel shape, with the chromophore inside. The chromophore is formed from residues serine 65 (S65), tyrosine 66 (Y66) and glycine 67 (G67). The process of creating the structure of the protein is called folding, whereas the process of creating the chromophore is termed maturation. Maturation occurs after the protein folds to near native conformation.

However, the natural, wild type, GFP is not the best tool. It is rather dim and not very stable at 37°C  (Since the jellyfish lives at low temperatures). Also, it matures rather slowly. Early on, a mutation was introduced, S65T, that increased the brightness of the GFP and also shifted its excitation peak (from a major peak at 395 and a minor at 475, to a single major peak at 488).  In vivo, it was shown that maturation of GFP-S65T takes 27 minutes (this was measured by expressing GFP in E. coli bacteria under anaerobic conditions, then supplementing the bacteria with O2. Therefore, they only measured the last few steps of the maturation process). EGFP, the most common variant used in research labs, contains a second mutation, F64L. This mutation stabilizes the protein at 37°C and was suggested to increase the maturation rate.

Over the years, GFP has been a subject for further “directed evolution” to achieve required traits like different colors, brightness, pH stability and photostability, oligomerization state, as well as folding and maturation.

Why do we care about folding and maturation?

Well, GFP is often fused to other proteins. In some cases, that protein is misfolding, or folding slowly. This can affect the folding of GFP. This is particularly true when expressing fused proteins in E. coli, where in many cases the ectopically expressed protein is aggregating in inclusion bodies. Since GFP itself has a slight tendency to dimerize, any aggregation of the fusion protein may amplify the dimerization of the GFP. Thus, it would be advantageous to have a better folding GFP, with a lower tendency to dimerize.

As to maturation, having a fast-maturing GFP would be very beneficial for studies of translation, or translation-coupled localization.  Because, if the GFP takes 20-30 minutes to mature after it is being translated, we cannot really say what happened and where was the GFP or any fused protein, in that time.

Our story begins in 1996, when the lab of Willem Stemmer tried to improve GFP by a technique called DNA shuffling. Their goal was to improve whole cell fluorescent in E. coli cells expressing GFP. After three cycles of DNA shuffling, and selection based on fluorescence intensity with UV excitation, they isolated a clone (named simply ‘GFP cycle 3’) which showed 45-fold increased fluorescence compared to the then commercial Clontech pGFP. However, the spectral characteristics were unchanged, and the maturation rate (T1/2 = 95min at 37°C in whole cells) also seemed unchanged).  Mutations found in GFP cycle 3 compared to their starting GFP showed that three hydrophobic amino acids were replaced by hydrophilic amino acids. Thus, the improved fluorescence is probably due to reduction in protein aggregation by the hydrophobic surfaces, and increased solubility of the GFP.

CHO cells expressing wt GFP (A) or GFP cycle 3 (B). Source: Crameri et al. (1996) Nat. Biotech. 14:315.

Ten years later, the superfolder GFP (sfGFP) was engineered by the lab of Geoffrey Waldo for the particular purpose of creating a protein with less tendency to misfold when fused to poorly folded proteins in E. coli.

How was this protein engineered?

They started with a folding reporter GFP  (frGFP) which contained the cycle 3 & EGFP mutations.

They fused frGFP to a poorly folding protein, H subunit ferritin, which is insoluble when expressed in E. coli. After several rounds of mutagenesis and screening for fluorescence, they obtained the highly fluorescent sfGFP which bears six new mutations.  Ferritin-sfGFP was found to be 50-fold more fluorescent than the starting fusion protein. When expressed alone, cells expressing sfGFP were twice as bright as with frGFP. Importantly, the excitation & emission spectra , QY and EC were similar in both proteins.

Measuring the folding rate was done by first denaturing the proteins with urea. Then washing the urea and measuring the time it takes the GFP variant to regain fluorescence.  This method actually measures folding and maturation and this should be taken into account. In any case, though both proteins showed 95% yield within 4 minutes, sfGFP showed a 3.5-fold faster initial rate. sfGFP also tolerated higher urea levels, and showed better fluorescence with a bunch of different fused proteins with different characteristics (expression level, solubility etc…).

fusion of frGFP or sfGFP to 18 differnt proteins shows sfGFP much brighter in all cases. 0.125s, 1s: exposure time. below the images of the bacteria are Western blot images of the fusion proteins. Source: Pedelacq et al. (2006). Nat. Biotech. 24(1):79

They found that the most contributing mutation to the folding  kinetics and stability is the S30R mutation. The change from an oxygen group (that forms a hydrogen bond with E17) to a positively charged side chain mediates an electrostatically charged network of the β-barrel which seems to create a global stability to the structure. A second mutation, Y39N, slightly changes the angle of the backbone, and allows a hydrogen bond with D36, further stabilizing the structure. The other four mutations slightly contribute to the stability.

Although sfGFP seems to fold fast (though no comparison to EGFP was made in this paper), the assays here did not really differentiate folding from maturation.

A year later, the group of David Liu used sfGFP  in an effort to create a new GFP protein that is highly soluble. In order to do that, they replaced multiple neutral amino acids on the outer side of the protein with either positively or negatively charged amino acids to produce super-charged GFP variants GFP(+36) and GFP(-30) [compared to the net charge of sfGFP which is (-7) or GFP which is (-8)]. This scGFP was highly soluble, and also highly resistant to denaturing, even by boiling to protein at 100°C. The spectral characteristics of the scGFPs remained similar to those of GFP and sfGFP indicating that the structure of the outside “barrel” does not affect the chromophore features inside the barrel.

In 2008, Fisher & DeLisa produced yet another variant of GFP called superfast GFP. By exploiting the cell’s protein secretion quality control mechanisms, they screened for new super folding variants. The secretion mechanism exports unfolded proteins to the periplasm of E. coli. In the periplasm, GFP is apparently non-fluorescent. If the protein folds fast enough, the quality control mechanism prevents its export and the protein remains in the cytoplasm.  Their starting GFP was GFPmut2, a variant that was previously optimized for FACS analysis, harboring the mutations S65A/V68L/S72A. Following several round of selection, they isolated a clone which they named superfast GFP. Based on their analysis (using guanidinium chloride (GdnCl) to unfold the proteins and then diluting the GdnCl to refold the proteins, the T1/2 for superfast GFP refolding (resumed fluorescence) was 11 minutes, compared to 33 min (GFPmut2), 20min (sfGFP) and 73min (frGFP).

Graph showing folding of GFP proteins over time. black: square – frGFP; circle – sfGFP; triangle – GFPmut2. open circle – superfast GFP; triangle, square – two other mutants. Source: Fisher AC & DeLisa MP. (2008). PLoS One. 3(6):e2351.

So, far we discussed fast folding GFP proteins. However, the methods to measure folding of these proteins do not discriminate folding from maturation.  Furthermore, the in vitro results do not necessarily represent the in vivo folding rates (i.e. 11 min in vitro does not mean 11 min in vivo).

In the next post I will discuss the issue of maturation.

ResearchBlogging.orgCrameri A, Whitehorn EA, Tate E, & Stemmer WP (1996). Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature biotechnology, 14 (3), 315-9 PMID: 9630892
Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, & Waldo GS (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature biotechnology, 24 (1), 79-88 PMID: 16369541
Lawrence MS, Phillips KJ, & Liu DR (2007). Supercharging proteins can impart unusual resilience. Journal of the American Chemical Society, 129 (33), 10110-2 PMID: 17665911
Fisher AC, & DeLisa MP (2008). Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control. PloS one, 3 (6) PMID: 18545653