The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult task for structure-function studies. Based on codon optimization for protein expression in Escherichia coli, we have been able to produce, in soluble form, the C-terminal domains of Inscuteable and Staufen as chimeras with N-terminal maltose binding protein tag that contains a rigid linker between them for feasible crystallization. In addition, using an optimized synthetic gene, corresponding to the amino acid region 250 - 623 of Inscuteable fused to glutathione-S-transferase, low-scale expression experiments showed production of soluble protein. Finally, eukaryotic expression of Inscuteable in the methylothropic yeast Pichia pastoris failed to produce the Drosophila protein at detectable amounts, reinforcing the fact that E. coli still was the microorganism of choice for high-yield protein expression.
Asymmetric cell division is of one nature’s processes to generate cellular diversity; in Drosophila melanogaster neuroblasts are considered the stem cells of the central nervous system [
The cell fate determinants are biomolecules that regulate differentiation between neuroblasts and GMCs, and the asymmetric localization of these determinants is the key for the proper development and lineage of GMCs [
Inscuteable is the most upstream component for the asymmetric division of neuroblasts and GMCs [
The understanding of asymmetric localization of cell determinants during cell division at the molecular level would be extremely useful in order to further comprehend development and cellular diversity from stem cells. Here we describe the expression and purification of the two domains of Inscuteable mentioned above and the C-terminal of Staufen for protein crystallization and structure-function studies. The task was undertaken with different methodologies in order to obtain several milligrams of protein for structural studies: from eukaryotic protein expression in the eukaryote Pichia pastoris to gene synthesis for codon and DNA secondary structure optimization for expression in Escherichia coli. We found that with the right construct E. coli is still the most suitable host microorganism for high-yield production of recombinant proteins.
The primary sequences of Staufen and Inscuteable were entered into the Primo Optimum 3.4 software (Chang Bioscience), which produced DNA sequences containing the codons most commonly used by E. coli. The program also designed the alternating oligonucleotides needed for the PCR synthesis of the gene [
NAME | PROTEIN | AA REGION SIZE§ | AFFINITY TAG | HOST CELL |
---|---|---|---|---|
Insc1 | Inscuteable | 719 - 859 15.3 kDa | GST* N-terminal | E. coli |
Insc2 | Inscuteable | 719 - 857 15.0 kDa | GST N-terminal | E. coli |
Insc3 | Inscuteable | 1 - 859 96.4 kDa | His-tag C-terminal | P. pastoris |
Insc4 | Inscuteable | 719 - 857 15.0 kDa | His-tag C-terminal | P. pastoris |
Insc5 | Inscuteable | 719 - 857 15.0 kDa | GST N-terminal | P. pastoris |
Insc6 | Inscuteable | 250 - 601 37.1 kDa | GST N-terminal | P. pastoris |
Stau1 | Staufen | 761 - 1026 27.7 kDa | GST N-terminal | E. coli |
Insc7 | Inscuteable | 719 - 857 15.0 kDa | GST N-terminal | E. coli |
Insc8 | Inscuteable | 719 - 857 15.0 kDa | His-tag N-terminal | E. coli |
Insc9 | Inscuteable | 250 - 623 41.6 kDa | GST N-terminal | E. coli |
Insc10 | MBP+/Inscuteable chimera | 719 - 857 55.9 kDa | MBP N-terminal | E. coli |
Insc11 | MBP/Inscuteable chimera | 719 - 859 57.2 kDa | MBP N-terminal His-tag C-terminal | E. coli |
Stau2 | MBP/Staufen chimera | 761 - 1026 68.6 kDa | MBP N-terminal | E. coli |
§Indicates the molecular weight without considering the removable affinity tag. For the chimeras, it includes the affinity tag(s). *Glutathione-S-trans- ferase. +Maltose binding protein.
For E. coli expression, the BL21(DE3) or Rosetta strains were used. For pilot protein expression experiments, 2 mL of LB medium (containing 100 μg/mL ampicillin) were inoculated with a single E. coli colony and incubated at 37˚C in a shaking incubator. During log-phase growth, expression was induced with 0.5 mM isopropyl- D-1-thiogalactopyranoside (IPTG). After induction, the temperature was lowered to 25˚C and the cells were incubated overnight. Cells were harvested and resuspended in 100 μL of SDS-PAGE sample buffer and boiled for 10 minutes. After centrifugation the supernatant was analyzed by electrophoresis. Large-scale protein expression was performed in baffled fernbach flasks using LB-amp medium, cells were incubated at 37˚C until O.D.600 reached 0.6. Then the temperature was lowered to 25˚C and IPTG was added to a final concentration of 0.5 mM. Cells were further incubated for 16 hrs. and harvested by centrifugation. For P. pastoris expression, the GS115 strain (Life Technologies) was transformed by electroporation with 10 μg of linear DNA and selection of transformants was performed using the manufacturer’s suggested protocol. For P. pastoris protein expression induction, cells were grown in minimal methanol medium in baffled fernbach flasks at 30˚C.
After cell disruption (sonication for E. coli and treatment with YeastBuster (Novagen) for P. pastoris) and clarification of lysates by centrifugation, proteins were purified by affinity column chromatography. For GST-tagged proteins, lysates were incubated with glutathione sepharose 4B in PBS buffer at room temperature for one hour. The resin was loaded into a column and washed several times with PBS buffer; proteins were eluted in three column-volumes using elution buffer: 50 mM TRIS-HCl, 10 mM reduced glutathione, pH 8.0. Purification of proteins with a His-tag was performed using the His-Pur Ni-NTA resin from Thermo Scientific following manufacturer’s instructions. Insc10 and Insc11 were further purified by anion exchange and size exclusion chromatography using the ÄKTA PrimePlus FPLC system from GE Life Sciences. After affinity chromatography, proteins were loaded into a MonoQ anionic exchange column in 50 mM TRIS-HCl buffer at pH 7.8. The column was washed extensively with the same buffer; a 0 - 250 mM NaCl gradient was applied in 40 column-volumes, the fractions that showed the higher protein concentration were analyzed by SDS-PAGE. The purest fractions were pooled and concentrated up to 240 μL, then loaded in a Superose 12 column for size-exclusion separation. Final protein purity was determined by SDS-PAGE.
The C-terminal domain of Inscuteable that interacts with Staufen for prospero RNA localization is reported to be the amino acid region 751 to 859 [
This kind of result is not uncommon for eukaryotic proteins expressed in E. coli [
Several DNA constructs were made for expression of Inscuteable in the yeast Pichia pastoris. Full-length Inscuteable DNA Insc3 and its C-terminal region Insc4, both with a C-terminal his-tag, were cloned into pPIC3.5K plasmid vector.
Because of unsuccessful attempts to amplify Staufen DNA from a cDNA library, a different approach was needed
to make the constructs for protein expression. PCR-based DNA synthesis is a method that allows rapid production of a nucleotide sequence optimized for expression in a certain system [
The newly synthesized Staufen DNA was cloned into pGEX-4T-2 vector for the Stau1 construct. After GST af-
finity chromatography the construct yielded several milligrams of protein. It was observed that the band appeared as a higher molecular weight protein than expected, so the protein was further characterized by mass spectrometry and sequencing. The mass spectrum showed a value of 54,287.06 m/z that is in good agreement with the calculated value of 54,051.90 Daltons.
Maltose binding protein is a well-known fusion protein that produces high quantity of protein in a soluble form in E. coli. A different study has shown that MBP can be a powerful tool for protein crystallization and structure determination [
order to minimize the flexibility between the two domains of the final protein [
Insc10 was purified by affinity chromatography with amylose resin. The pooled fractions containing Insc10 were dialyzed and loaded into a MonoQ anionic exchange column. After several column-volume washes, the protein was eluted with a NaCl gradient. The two purest fractions were pooled, the protein concentrated and then applied to Superose 12 column for size-exclusion chromatography. This was a final purification step and it also helped to determine the oligomeric state of Insc10. Based on the elution volume (11.8 mL), it appeared that Insc10 is a monomer; it is worth mentioning that MBP is a monomer and it has been observed that its presence does not affect the quaternary structure of the other protein in the chimera [
Fractions 3 and 4 after size-exclusion chromatography of Insc10 and pure Insc11 were concentrated up to 15 mg/mL for protein crystallization trials, and showed good solubility at that concentration. Crystallization could be feasible due to the designed linker between MBP and the C-terminal of Inscuteable [
The generation of cellular diversity in developing organisms requires the synchronization of several molecular mechanisms; in Drosophila central nervous system, Inscuteable and Staufen play an important role for the asymmetric localization of cell fate determinants. This work provides a simple process for the production of large quantities of these proteins in E. coli for future structure-function studies. Gene synthesis was demonstrated to be a powerful tool for the expression of the Drosophila proteins Inscuteable and Staufen. The chimera design with maltose binding protein helped considerably to express high amounts of the C-terminal of both proteins, while the fusion protein glutathione-S-transferase allowed the soluble production of the main functional domain of Inscuteable.
XristoZárate,Megan M.McEvoy,TeresaVargas-Cortez,Jéssica J.Gómez-Lugo,Claudia J.Barahona,Elena Cantú-Cá rdenas,Alberto Gómez-Treviño, (2015) Purification of the Drosophila melanogaster Proteins Inscuteable and Staufen Expressed in Escherichia coli. Advances in Bioscience and Biotechnology,06, 485-493. doi: 10.4236/abb.2015.67050