This work was supported by the new faculty startup funds from North Carolina State University for Guozhou Xu.

NOTE: An insect cell/baculovirus system with modified expression vectors for secretory plant protein expression and crystallization is used.
1. Modification of a Baculovirus Expression Vector with the GP67 Signal Peptide for Plant Protein Secretion Expression
<ol>
	<li>Synthesize a DNA fragment containing a 5&#39; BglII cutting site13, the GP67 secretion signal sequence, and a multi-cloning site with NotI, BamHI, EcoRI, StuI, SalI, SpeI, XbaI, PstI, and XhoI (<strong ...

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Given the diversity in size and stability of the thousands of proteins present in the biological systems, it is often empirical for a research laboratory to decide which heterologous expression system has to be chosen for the expression of a specific protein. The E. coli expression system is often the first choice for protein expression due to the short life cycle of the bacteria, low cost of the culture media, and relative ease to scale up19. For the expression of large eukaryotic protei...

It is imperative for a research laboratory to be capable of producing large quantities of homogeneous recombinant proteins for biochemical and biophysical characterizations, especially for X-ray crystallographic studies. There are many well-established heterologous expression systems such as Escherichia coli, yeast, insect cells, mammalian cells, plant cells, etc. Among them, the baculovirus-mediated insect cell expression system is one of the most commonly used techniques to produce large quantities of structurally folded large-sized recombinant eukaryotic proteins for protein crystallization1.
The expression vectors of the baculovirus expression system are engineered to contain a strong polyhedrin or P10 promoter to produce a high yield of recombinant intracellular proteins2,3. To make a recombinant baculovirus, the gene of interest is cloned into an insect vector containing the polyhedrin (polh) locus of the Autographa californica multi-nucleopolyhedroviral genome. The resulting construct is then sequenced and its correct open reading frame (ORF) is verified. The correct construct is then introduced into the host insect cell through the process of transfection. The gene of interest is inserted into the viral genome by homologous recombination. This event results in the production of the recombinant viral genome, which then replicates to produce recombinant budded virus particles1.
The insect cells that are most commonly used in the expression system are Sf9 and High Five (Hi5) cells. Sf9 cells are a clonal isolate of Sf21, derived from the pupal ovarian cells of Spodoptera frugiperda, and Hi5 cells are a clonal isolate derived from the parental Trichoplusia ni ovarian cell line TN-3684,5. Co-transfections, virus amplification, and plaque assays are conducted on Sf9 cells, while Hi5 cells are typically selected to produce higher quantities of recombinant proteins6. It is worth noting that the Hi5 cells are not suitable for the generation and amplification of virus progenies because of their tendency to produce mutant viruses. Traditionally, a temperature range of 25 - 30 &#176;C is considered to be good for the cultivation of insect cells. However, it has been reported that 27 - 28 &#176;C is the optimal temperature for the insect cell growth and infection7,8.
The introduction of a strong signal sequence preceding the gene is needed for the high expression of the secreted proteins. The signal sequence would efficiently guide the translated recombinant protein into the endoplasmic reticulum for protein secretion and post-translational modifications necessary for proper folding and stabilization3. Signal peptide sequences, such as the baculovirus envelop protein GP64/67, honeybee melittin, and others, have been chosen to facilitate the expression of secretory recombinant proteins in the baculovirus-mediated expression systems3. The introduction of the signal peptide of GP67 has been shown to improve the expression yield of a secreted recombinant protein, in comparison to using the intrinsic signal peptide of the target gene9. Hemolin is a hemolymph protein of the giant silk moth Hyalophora cecropia, induced upon bacteria infection10. Due to the relatively high level of induced expression, the signal peptide sequence of the gene can be used to mediate the secretion expression of the recombinant proteins in the baculovirus-insect cell system.
The A. thaliana Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and Pollen Receptor Kinase 3 (PRK3) both belong to the plant Leucine-rich Repeat Receptor-like Kinase (LRR-RLK) family of proteins11,12. In order to study the structure and function of this family of plant receptor proteins, as well as to facilitate the structural and biochemical characterization of other plant secreted proteins, the baculovirus-insect cell expression system has been modified to improve protein quality and production yield. The extracellular domains of both TDR and PRK3 have successfully been expressed using two modified expression vectors in the baculovirus-insect cell expression system. Both the extracellular domains of TDR and PRK3 proteins have been crystallized. This article reports the expression and purification of large amounts of recombinant secreted plant proteins with two modified baculovirus expression vectors by incorporating either a GP67 or a hemolin signal sequence between the promoter and multiple cloning sites.

<table>
	<tbody>
		<tr>
			<td>Incubator shaker</td>
			<td>VWR</td>
			<td>Model Excella E25</td>
			<td>27 oC</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model 2005</td>
			<td>27 oC</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>BECKMAN</td>
			<td>Model J-6</td>
			<td>with a swing bucket rotor</td>
		</tr>
		<tr>
			<td>Herculase II Fusion DNA Polymerase</td>
			<td>Agilent</td>
			<td>600677-51</td>
			<td>For PCR amplification of DNA</td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>BIO-RAD</td>
			<td>Model C1000 Touch</td>
			<td>For PCR amplification of DNA</td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Model I 24</td>
			<td>For growing baceria culture</td>
		</tr>
		<tr>
			<td>Customer DNA synthesis</td>
			<td>GENSCRIPT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI (HF)</td>
			<td>New England Biolabs</td>
			<td>R3136S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>New England Biolabs</td>
			<td>R0144S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>NotI (HF)</td>
			<td>New England Biolabs</td>
			<td>R3189S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Restriction Endonuclease</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td>DNA ligation</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>DNA purification from Agorase gel</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>Plasmid DNA purification from bacteria culture</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>15510-019</td>
			<td>For DNA gel electropherosis</td>
		</tr>
		<tr>
			<td>MAX Efficiency DH5&#945; competen cell</td>
			<td>Invitrogen</td>
			<td>18-258-012</td>
			<td>For transformation of DNA ligation mixture</td>
		</tr>
		<tr>
			<td>Lennox L LB Broth</td>
			<td>Research Product International Corp.</td>
			<td>L24066-5000.0</td>
			<td>For making bacteria culture</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Thermo Fisher Scientific</td>
			<td>11593-019</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>15160-054</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Thermo Fisher Scientific</td>
			<td>64-75-5</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15710-064</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>MAX Efficiency DH10Bac competent cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>10361-012</td>
			<td>For making bacmid DNA</td>
		</tr>
		<tr>
			<td>S.O.C. Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>15544-034</td>
			<td>For DNA transformation</td>
		</tr>
		<tr>
			<td>CellFECTIN II Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362-101</td>
			<td>Insect cell transfection reagent</td>
		</tr>
		<tr>
			<td>Bac-to-Bac Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>10359-016</td>
			<td>Baculovirus-insect cells expression kit</td>
		</tr>
		<tr>
			<td>Bluo-gal</td>
			<td>Thermo Fisher Scientific</td>
			<td>15519-028</td>
			<td>For isolation of recominant Bacmid DNA</td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Fisher Scientific</td>
			<td>15529-019</td>
			<td>For isolation of recominant Bacmid DNA</td>
		</tr>
		<tr>
			<td>pFastBac1</td>
			<td>Thermo Fisher Scientific</td>
			<td>10360014</td>
			<td>Baculorirus expression vector</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>11496015</td>
			<td>Sf9 monolayer cells</td>
		</tr>
		<tr>
			<td>Hi5 cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>B85502</td>
			<td>High Five insect cells</td>
		</tr>
		<tr>
			<td>Grace&#8217;s insect medium, unsupplemented</td>
			<td>Thermo Fisher Scientific</td>
			<td>11595030</td>
			<td>Sf9 cell transfection minimum medium</td>
		</tr>
		<tr>
			<td>Grace&#8217;s insect medium, supplemented</td>
			<td>Thermo Fisher Scientific</td>
			<td>11605102</td>
			<td>Sf9 monolayer cell culture complete medium</td>
		</tr>
		<tr>
			<td>Sf-900 II SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10902104</td>
			<td>Sf9 suspension cell culture medium without FBS</td>
		</tr>
		<tr>
			<td>Express Five SFM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10486025</td>
			<td>Hi5 cell culture medium</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 ml (10,000 I.U./ml)</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>FBS Certified</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-772-1B</td>
			<td>Flat-bottom</td>
		</tr>
		<tr>
			<td>150 mm plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>353025</td>
			<td>100/case</td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1415-2500</td>
			<td>500 tubes/bag</td>
		</tr>
		<tr>
			<td>15 ml conical screw cap centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1475-0511</td>
			<td>25 tubes/bag</td>
		</tr>
		<tr>
			<td>50 ml conical screw cap centrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1500-1211</td>
			<td>25 tubes/bag</td>
		</tr>
		<tr>
			<td>Ni-NTA Superflow</td>
			<td>Qiagen</td>
			<td>30430</td>
			<td>NiNTA resin</td>
		</tr>
		<tr>
			<td>pH-indicator strips</td>
			<td>EMD Millipore Corporation</td>
			<td>1.09535.0001</td>
			<td>pH 0 - 14</td>
		</tr>
	</tbody>
</table>

modifying baculovirus expression vectors to produce secreted plant proteins in insect cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

As shown in Figure 1, two modified pFastBac1 baculovirus expression vectors were used to express the secreted proteins with either the GP67 or the hemolin signal sequence to replace the intrinsic signal sequence of the target gene. The viral GP67 and the insect hemolin genes have been demonstrated to have high secretion expression levels in the cells. Fusion proteins with either of these two signal sequences are expected to have greatly improved secretion exp...

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Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

This method can help answer key questions in the biochemistry, and structural biology field of secreted proteins, such as protein structure determination of, by crystallography and cryo-EM. The main advantage of this technique is it produces large amount of recombinant secreted proteins with relatively low cost for protein structure determination. First, synthesize a DNA fragment containing a five prime BglII cutting site, the secretion signal sequence of interest in a multi-cloning site as outlined in the text protocol.Next, digest four micrograms of the DNA with BglII and X1, and four micrograms of an expression vector DNA with BamH1 and X1.Mix 10 units of each restriction enzyme with the DNA in an 1X concentration reaction buffer. Incubate at 37 degrees Celsius for four hours. Add 10 microliters of a ligation reaction mixture containing 1X T4 DNA Ligase reaction buffer and five units of T4 DNA Ligase to a fresh 1.5 milliliter Eppendorf tube.Then, add 100 nanograms of the linearized vector DNA and 400 nanograms of the digested DNA fragment. Incubate at 16 degree Celsius for 16 hours. Transform five microliters of the ligation mixture into 100 microliters of DH5 alpha competent cells and select the resulting colonies on an Ampicillin LB agar plate as outlined in the text protocol.Pick between five and 10 colonies transferring each to two milliliters of LB medium containing 100 micrograms per milliliter ampicillin. Grow these colonies in a shaking incubator at 220 rpm in 37 degree Celsius for 16 hours. After this, use a DNA Miniprep kit to extract each plasmid DNA.Confirm the clones by DNA sequencing with a forward sequencing primer as outlined in the text protocol. Using 100 nanograms of the construct plasmid, transform 40 microliters of DH10Bac competent cells as outlined in the text protocol. Incubate the transformation plates at 37 degrees Celsius for 48 hours.Pick up two white colonies from each transformation plate and inoculate each colony in two milliliters of LB medium containing kanamycin, gentamycin, and tetracycline. Extract and verify the bacmid DNA as outlined in the text protocol. Aliquot each DNA into two tubes with 20 microliters being transferred to each tube.Then store the tubes at minus 20 degree Celsius until ready to proceed. All subsequent steps must be performed aseptically. In a six-well plate, grow a monolayer of Sf9 cells in the culture media with 10%fetal bovine serum, and 100 micrograms per milliliter Penicillin-Streptomycin antibiotics at 27 degrees Celsius to 80%confluence.For each transfection, add 100 microliters of unsupplemented Sf9 cell culture medium without antibiotics to a 1.5 milliliter centrifuge tube. Dilute 20 microliters of bacmid DNA into each tube and gently flick to mix. Next, add 100 microliters of unsupplemented Sf9 cell culture medium without antibiotics to a 1.5 milliliter tube for each transfection.Dilute eight microliters of lipid transfection reagent into each tube, and mix by thoroughly pipetting up and down six times. Combine these two solutions and mix them by gently pipetting slowly up and down three times. Incubate for 20 to 40 minutes at room temperature.Wash each well of the culture plate twice using three milliliters of unsupplemented Sf9 culture medium without antibiotics for each wash. Next, add 0.8 milliliters of unsupplemented Sf9 cell culture medium without antibiotics to each tube containing the lipid DNA mixture. Slowly pipet up and down three times to mix.Aspirate the wash media from the plates, and overlay the samples with the transfection mixture. Incubate the transfected plate for five hours at 27 degrees Celsius. After this, replace the transfection media with the complete Sf9 cell culture medium which contains 10%FBS, and 100 micrograms per milliliter Penicillin-Streptomycin antibiotics.Incubate at 27 degree Celsius for four days. Then, collect the supernatant in a sterile 15 milliliter conical tube. Spin at 1, 010 times g for five minutes to remove the cell debris.Discard the pellet, and decant the supernatant to a clean tube. Store at four degrees Celsius for up to one year or until ready to use. To amplify each recombinant virus, first grow a monolayer of Sf9 cells on a 150 millimeter plate to 80%confluence.Then aspirate the medium, and add two milliliters of the P0 virus to each well. Incubate for one hour in a 27 degree Celsius incubator, rocking the plate every 15 minutes to mix the medium with the cells. Next, add 25 milliliters of complete Grace's Media containing 10%FBS, and 100 micrograms per milliliter Penicillin-Streptomycin antibiotics.Incubate the plate for three days in a 27 degree Celsius incubator to obtain the P1 passage virus. After this, collect the supernatant in a sterile 50 milliliter conical tube. Spin at 1, 010 times g for five minutes to remove the cell debris.Decant the supernatant to a clean tube, and store at four degrees Celsius for up to one year or until ready to use. To amplify the virus from P1 to P2 passage, first grow a monolayer of Sf9 cells on a 150 millimeter plate to 90%confluence. Once the desired confluence is reached, aspirate the medium.Add two milliliters of the P1 virus and incubate for one hour in a 27 degree Celsius incubator. Repeat the amplification steps to obtain the P2 and P3 passages of the virus. In this study, two modified pFastBac1 baculovirus expression vectors are used to express the secreted proteins with either the gp67 or the hemolin signal sequence by replacing the intrinsic signal sequence of the target gene.The PBEC gp67 is successfully utilized to express the extracellular domains of the a-thal-leano receptor TDR. While the PBEC1 hemolin is successfully utilized to express the extracellular domain PRK3. Next, the purified proteins are concentrated to five milligrams per milliliter, and subjected to crystallization screening.The conditions which yielded preliminary crystals for each protein are optimized until protein crystals larger than 20 micrometers are observed. While attempting this procedure, it's important to remember they examine the quality of the proteins by size exclusion chromatography, multi-angle light scattering, and other assays for non-aggregation status of the recombinant protein. Don't forget, to check the purity of the protein also by silver stain.Following this procedure, other methods like X-ray crystallography and cryo-EM can be performed in order to answer additional questions, like the three dimensional structures of the produced protein. After its development, this technique paved in the way for researchers in the field of structural biology to explore the structural, and functional, and biochemical properties of secreted proteins from plants and in mammals. Don't forget that working with ultraviolet light to visualize the DNA bands on the agarose gel can be extremely hazardous, and precautions such as wearing UV protections, face shield, eye goggles, lab coat, should always be taken while performing this procedure.Also while making buffers, be careful while handling strong acids and bases.

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Biochemistry

11.3K visualizações.  North Carolina State University. This method can help answer key questions in the biochemistry, and structural biology field of secreted proteins, such as protein structure determination of, by crystallography and cryo-EM. The main advantage of this technique is it produces large amount of recombinant secreted proteins with relatively low cost for protein structure determination. First, synthesize a DNA fragment containing a five prime BglII cutting site, the secretion signal sequence of interest in a multi-cloning site as o...