The HTP Process

Rational Project Design | Ligation Independent Cloning (LIG) | Transformation | Expression | Lysis | Purification | Assay

Rational Project Design

Assemble background information about a project protein class (kinase, cytokine, receptor, etc.), previous expression data, functional domain information, downstream application.

Determine predicted folding domains using programs such as Foldindex and DisEMBL. These are server based programs available to the public. Overlay this information with functional domain information and secondary structure prediction. Design constructs using this synthesis. Examine codon usage and perform codon optimization if necessary.

Oligos for PCR construction are designed using Clone-manager. Oligos are optimized for a number of parameters; reduction of primer dimers, hairpins, false priming sites, annealing temperature, etc. This may require the introduction of silent mutations. Oligos are designed to incorporate LIC cloning sites compatible with the vectors sets in use in the HTP.

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Ligation Independent Cloning (LIC)

Ligation independent cloning is similar to traditional cloning, but with differences in how the overhangs are created and how the gene of interest is inserted into the vector. The process both increases the success rate and decreases time and reagent costs.

Steps for LIC:

• Long primers are used in the PCR construction of inserts that extend the ends of the product, which will be cloned into a LIC vector.
• T4 DNA polymerase has a 3'->5' exonuclease activity that predominates over polymerase activity when no nucleotides are present for polymerization. When dCTP is present with the PCR product, T4 DNA polymerase will remove nucleotides until reaching a G in the template strand, then stop, creating a 15 nt overhang.
• The PCR product with the overhangs is combined with a LIC vector that has gone through the same process with dGTP to create complimentary overhangs. The vector and PCR product are annealed and then transformed into XL1 blue cells.
• The XL1 blue cells will ligate the breaks in the plasmid and due to the presence of a selective marker; the plasmid will be established and replicated. The plasmid can then be purified and transformed into expression strains.

There are a number of vectors that the PCR product can be cloned into that contain identical overhangs. These vectors have various tags such as 6X His, and MBP to aid solubility and purification. The vectors in use in the HTP lab are modified versions of pMCSG7 and pMCSG9. Etti Harms at Purdue University constructed these variants.

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Transformation

Cell line is one of the parameters that have been found to have a drastic impact on expression levels in some cases. A number of different cell lines are available for expression testing.

Some cell line examples:

• BL21-AI, arabinose inducible cell line: The chromosomal copy T7 RNA polymerase is under the control of an arabinose inducible promoter (araBAD). This leads to increased control of gene expression, which is especially useful for toxic proteins.
• BL21-Rosetta and Rosetta 2: Many eukaryotic genes have codons that are low usage in E. coli. This leads to low expression level. To compensate for this the gene may be codon optimized or a cell line containing a plasmid with copies of the low use tRNAs may be used. These cell lines are named Rosetta and the plasmids they contain are pRARE1 or pRARE2. They vary in the number of additional tRNA genes that they carry.
• Origami: This cell line is designed to provide enhanced disulfide bond formation in the cytoplasm. The E. coli cytoplasm is a highly reducing environment and disulfide bonds are rarely found in heterologously expressed proteins. Mutations in thioredoxin reductase (trxB) and glutathione reductase (gor) enhance disulfide bond formation, which is useful for proteins that require disulfide bonds.
• BL21-pLysS: The pLysS plasmid encodes a constitutively expressed copy of the T7 lysozyme gene. T7 lysozyme inhibits T7 RNA polymerase. This leads to lower levels of protein expression, and possible increases in protein solubility. This also causes these cells to be rather fragile and lysis may be an issue in their handling.

Other cell lines include C41, C43, BL21-Star, Turner, etc.

Co-transforming chaperones

Chaperones are proteins that aid in protein folding. Coexpression of chaperones has been shown to increase soluble protein recovery.

Some Chaperone examples:

• dnaK, dnaJ, GrpE: These proteins maintain nascent proteins in prefolded states to help prevent aggregation and degradation. Once proteins are released, they can either finish folding on their own, or with the assistance of other chaperones.
• GroES, GroEL: These proteins work together and assist protein folding by binding exposed hydrophobic surfaces of unfolded or misfolded proteins and gives them 15 to 30 seconds in a protected environment to allow folding to occur. GroE assisted folding is most useful for protein under 60kDa.
• Trigger Factor: A chaperone-like factor that has been associated with GroEL substrate binding.

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Expression

Expression of constructs is performed in a 96 well block. Duplicates of these blocks can be grown under varying conditions to attempt to find which condition produces soluble expression.

Temperature: 15C to 37C are normally used

Media: ZYM5052(autoinduction media), LB, Minimal media, etc.

Inducer Concentration: 50uM to 1mM IPTG

Additives: Cofactors for the protein, desoxyglucose(non-metabolizable carbon source) can be added to the media.

Stress response: Variations in Oxygen levels, 6% EtOH.

Time: Short inductions of 1 to 4 hours can lead to increased levels of soluble protein whereas longer inductions can increase the total amount of protein.

Formatting and inoculation of the blocks is carried out using the Biomek liquid handler. Expression is done in 0.5 ml of media in a 96-well deep well block. Incubation is done in a New Brunswick Innova shaking incubator. In rich medias, these conditions allow the cultures to reach 14-17 OD 600, which is comparable to fermentation conditions and somewhat better than shake-flask. Suggestions for replicating these conditions on scale-up will be provided to downstream users. In our hands both growth and expression have shown good reproducibility with minimal well to well and plate to plate variation.

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Lysis

The lysis method and buffers can have a large effect on the solubility of the target protein.

Our basic lysis method involves chemical lysis of the cells directly in the media followed by separation of soluble and insoluble fractions by centrifugation. The plate format does allow cells to be pelleted and the media removed allowing the cells to be resuspended in any buffer.

The lysate is then transferred to automation friendly individual tubes and centrifuged at 20,000 x g. Complete separation of the soluble and insoluble fractions is produced.

We are developing a lysis buffer matrix to examine the effect of; low polarity co-solvents, kosmotropic salts, and pH. We will be testing 168 different combinations and this matrix will be available for use with proteins that continue to display low solubility in a wide variety of expression conditions.

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Purification

Purification can be performed simultaneously on all 96 soluble fractions after lysis. A 96 well filter plate is used which allows for automated washing of the resin and elution. The process is compatible with a number of affinity resins including Ni-NTA, amylose, and Glutathione Sepharose.

The purpose of the step is to remove contaminants and concentrate the protein of interest. This allows proteins with lower expression levels to be concentrated to the point where they can be viewed on gels.

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Assay

Determining if any of the previous strategies helped in producing soluble protein is the final step. We use the LabChip 90 automated electrophoresis system to analyze our soluble, elution, and insoluble samples. The system is capable of running 96 samples in approximately 90 minutes. The data is captured electronically where it can be analyzed in more depth than gels.

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