Chem/BMSE 259 Molecular Biology and Protein Purification Graduate Laboratory

Instructors:

Prof. Rick Dahlquist
Office: Chem 1126C, Phone: 893-5326
E-mail: dahlquist@chem.ucsb.edu, Website: http://www.chem.ucsb.edu/people/faculty/dahlquist/index.shtml

Prof. Deborah Fygenson
Office: Broida 2409, Phone: 893-2449
E-mail: deborah@physics.ucsb.edu, Website: http://www.physics.ucsb.edu/~deborah

Dr. Kalju Kahn
Office: PSB-N 2623, Phone: 893-6157
E-mail: kalju@chem.ucsb.edu, Website: http://www.chem.ucsb.edu/~kalju

Teaching Assistants

Rob Levenson, e-mail: rlevenson@chem.ucsb.edu
Tetsuya Kawamura, e-mail: tkawamura@chem.ucsb.edu
Anh Vu, e-mail: tkawamura@chem.ucsb.edu
Office: Chem 1317, Phone: 893-5468

Mission statement

The UCSB summer biochemistry lab course is designed to introduce participants whose primary field is not biochemistry to modern techniques of molecular cloning, mutagenesis, and protein purification. Course participants will carry out a project similar to that performed in many contemporary biochemistry and molecular biology laboratories. In the first part of the project, participants clone a gene for a protein involved in bacterial chemotaxis into a modern expression vector and change specific amino acids in this protein by site-directed mutagenesis of the corresponding plasmid DNA. In the second part of the project, participants express the wild-type and mutated proteins in the bacterial cells and purify the overexpressed proteins by means of column chromatography. In the third part of the project, participants characterize the wild-type and mutant proteins by electrospray mass spectrometry and perform binding studies using fluorescence and nuclear magnetic resonance spectrometry.

Experiments

The theory manuals and chapters of the operations manual can be downloaded here in the PDF format. Please note that you can follow hyperlinks that are in the PDF files by clicking on the link.

Instrumentation Manuals

Many experiments in this course involve use of materials or equipment for which manufacturers have provided nicely illustrated operations manuals. Normally, you want to obtain the most up-to-date versions of these manuals from the manufacturer's website. Copies of some of these documents are provided here for your convenience.

Scientific Publications of Interest

Student Files: Summer 2007

Practice: pBR322 or pGEM9?

• 6587
• 6595
• 6589
• 6593
• 6594
• 6596

Participants in the UCSB Summer Biochemistry Lab Course clone, mutate, purify, and characterize mutant forms of the CheY protein. This protein is an important regulator of bacterial chemotaxis. During the first week, participants sub-cloned the cheY gene from the pCW plasmid into the pET-28b vector in order to create a His-tagged recombinant protein. During the second week, the recombinant protein was purified using Ni-affinity chromatography. During the third week, mutant forms of CheY were created using PCR-based site-directed mutagenesis technology and the expressed proteins were characterized by mass spectrometry. The binding of FliM peptide to CheY was characterized by fluorescence spectrophotometry in order to determine the ionic strength dependence of the binding constant.

Description	Image
PCR Cloning of CheY One of the first steps in subcloning the cheY gene involves PCR-amplification of the gene out from the parent pCW plasmid. Two primers, complementary to the 5' and 3' ends of the cheY gene were designed such that the primers introduced a Nco I restriction site at the N-terminus of the protein and eliminated a stop codon at the C-terminus. The PCR product was separated from the template using agarose gel electrophoresis, extracted from the gel and digested with Nco I and Xho I restriction endonucleases. The image on the right shows lanes with the PCR product and Novagen's 100 bp marker in 1.2% agarose gel stained with the SYBR Safe dye. Other student images can be found here.
PCR Cloning of CheY The cheY> fragment was purified from the restriction enzymes by capturing the plasmid DNA into a microspin column followed by elution with water; this procedure typically yielded 150-250 ng of insert DNA. The first ten lanes in the image on the right show purified digested PCR products from participants in the course along with the 100 bp marker ladder. The other lanes show digested pET28 vectors. About 30 ng of insert DNA were loaded into narrow lanes and separated in 1.5% agarose gel until the Orange G dye reached the gel edge. The gel was stained for 45 minutes with the SYBR Gold stain and visualized using Safe ImagerTM via its orange cover. Image of the same gel taken through a yellow filter is here; an image obtained with the UV transilluminator (312 nm) is here.
Preparation of pET-28b pET-28(b) expression vector was used for subcloning the cheY gene into a system that allows expression of His-tagged CheY protein. The pET28(b) plasmid was linearized by treating 2000 ng of plasmid with NcoI and XhoI restriction endonucleases for three to four hours. The linearized fragment that contains the kanamycin resistance gene and the 6X-His tag was gel-purified from unreacted and partially reacted plasmids by gel electrophoresis. The image on the right shows BioRAD's 1 kb marker, a lane of undigested plasmid, and four lanes successfully digested pET28 fragment. Other student images can be found here.
Ligation of CheY with pET-28b The purified fragment of DNA containing the cheY gene was ligated into the linearized pET-28 vector. The ligation reaction contained about 20 ng of CheY fragment and 80 ng of linearized pET vector. The image on the right shows the DNA composition of the ligation reaction after incubation with T4 DNA ligase. Successful ligation should produce a slower-migrating circular DNA. While this gel; is difficult too interpret; it appears that many ligation reactions also yielded plasmid dimers. Due to the small quantity of DNA analyzed, it is possible that successful ligation products were present in samples that did not show the presence of band due to circular recombinant DNA.
Bacterial Transformation Competent E. coli (Stratagene XL10-Gold strain) were transformed with the ligation product using the heat-shock method. Transformed bacteria were plated on kanamycin-containing agar plates to select for clones that incorporated the ligation product. The image on the right shows one such agar plate after 16-hour incubation at 37 degrees. Note that not all the colonies have incorporated the cheY gene as infrequent self-ligation of the pET-28 also produces antibiotic-resistant bacteria. Other images of plates with cells transformed with ligation reactions or control reactions with a pET28 vector alone can be found here.
DNA Analysis The presence of the CheY gene in the recombinant plasmid in some samples was verified by the PCR. Primers complementary to the T7 promoter and the T7 terminator were used to amplify the insert and the PCR product was separated from the template in 1.2% agarose gel. The image here shows six successful recombinat plasmid samples that appear as intense bands at about 570 bp. Negative control due to the undigested pET28 (near the right edge) yields a 320 bp band. Also visible are faint bands due to the plasmid template in 5 kb region. The left lane contains 100 bp ladder. Gel mages from other participants can be found here.
Bacterial Transformation Purified plasmid with the CheY insert was used to transform BL21(DE3) cells using the heat-shock method. The bacteria that had successfully taken up the recombinant pET-28 vector were selected on kanamycin-containing agar plates (image on the right). A single colony was picked from this plate and transferred to 5 mL of sterile LB with kanamycin. Subsequent analysis confirmed that this colony indeed expressed the CheY protein. Plate images from other participants can be found here. Note that not every colony visible in these plates expresses CheY, for example a colony from another plate (IMG_0651) that only had four colonies was found to be devoid of CheY.
Protein Expression Analysis The expression of the CheY protein in the liquid bacterial culture was induced by adding 0.4 mM IPTG to the small liquid culture. After several hours, the cells were pelleted and lysed. The protein expression was checked by analyzing the protein composition using SDS polyacrylamide page electrophoresis. Unfortunately, the recombinant His-tagged CheY migrates nearly at the same rate as lysozyme, which was used for cell lysis, and the resolution of these two small proteins was unsuccessful in 12.5% polyacrylamide gel. Two samples were selected based on the relative band intensities for further analysis; subsequent analysis of lysate from larger cultures with 20% gel revealed that one of these samples expressed CheY.
Protein Purification Cells expressing the CheY protein in large liquid cultures were lysed and the cell lysate was fractionated by adding ammonium sulfate to 65% saturation. The protein pellet was redissolved and dialyzed against 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8. The His-tagged CheY protein was bound to nickel-NTA agarose resin and eluted with a gradient of imidazole. The purity of eluted fractions was determined by vertical SDS-PAGE. The image on the right shows one Coomassie-stained SDS gel in which the left lanes contain cell extracts before chromatography, and the following lanes contain various fractions. The first lane has molecular weight markers. SDS-PAGE images from other participants can be seen here.
Protein Immunodetection The presence of CheY in fractions was probed by a sensitive assay similar to immunodetection. Crude cell extract, flow-through, and fractions from the column were blotted to nitrocellulose membrane and probed with the Ni-NTA that was conjugated to horseradish peroxidase (HRP). The Ni-NTA binds to His-tagged proteins, and HRP catalyzes the formation of an insoluble purple dye at these locations. Thus, samples containing His-tagged proteins will yield a purple dot on the paper. The image on the right shows one such dot blot indicating that CheY eluted in fractions 5-11. Other imunoblots can be seen here.
Site-Directed Mutagenesis Few point mutants of CheY were created by changing the amino acids in the N-terminal region using the Stratagene QuikChange technology. The amplified double-nicked plasmid product was analyzed on the agarose gel after Dpn I treatment. The image on the right shows one such gel. The outermost lanes contain 1 kb size markers (NEB, 3 kb is bright), the other lanes have mutagenesis products. A successful mutagenesis is evident by the presence of a sharp band at about 6 kb (the double-nicked circular plasmid travels slower than the linear markers). Smear in lanes 8 and 9 suggest suboptimal reaction conditions (Pfu Ultra II enzyme with Pfu Ultra buffer?); low levels of primers in lanes 3 and 12 indicate primer degradation or incorrect dilution.
Sequencing of Mutant DNA Mutated DNA was purified from XL10-Gold or BL21-Gold cells using Stratagene Miniprep Kit and eluted in 50 microliters of water. UV-Vis spectrophotometric analys indicated that the plasmid DNA concentration in these samples was about 40-70 ng/microliter. About one microgram of plasmid DNA for each mutant was sent for sequencing to Northwoods DNA, Inc. Two of the samples (KK4 and KK7) yielded reasonable quality sequence data confirming the presence of CheY in the recombinat DNA. The results shown on the right confirm that an S15A mutant was created based on the wild-type CheY sequence in sample KK4. Another sequencing run confirmed that the lysine codon at position 13 in the triple mutant was successfully reversed to the wild type glutamic acid codon.
Transformation with Mutant DNA The competent E. coli cells (XL10 Gold or BL21(DE3)-Gold strain) was transformed with the product of the site-directed mutagenesis reaction. Transformed bacteria were plated on kanamycin-containing agar plates to select for clones that took up the recombinant DNA. We observed that plating transformed cells directly onto plates with IPTG reduces the viability of the E. coli dramatically. Note that the presence of colonies in this experiment merely indicates that the bacteria had acquired resistance for kanamycin; the clones may or may not express His-tagged CheY or may arise from transformations with the remaining template plasmid that expresses wild-type CheY. Other mutants are here.
Colony Expression Analysis The expression of four mutant forms of CheY was assayed by blotting the colonies onto nitrocellulose paper and inducing protein expression by growing blotted colonies on agar plates containing 0.25 mM IPTG. The membrane-bound cells were lysed and the presence of CheY was detected with Ni-NTA-HRP conjugate. This assay proved inconclusive, possibly due to overly large colonies that were difficult to lyse. Thus, the protein expression was re-tested via SDS-PAGE of lysates from liquid cultures of cells that presumably expressed one of the mutants. The image on the right illustrates that all but one of the mutant CheY transformations was successful.
Purification of Mutant CheY A colony expressing double mutant CheY was picked from the plate above and used to grow 600 mL liquid culture. Protein expression was induced by adding 1 mM IPTG when the absorbance at 600 nm had reached about 0.5. Bacteria were lysed using freeze-thawing, and the His-tagged mutant CheY was purified from lysate with Ni-NTA spin columns. The cell extracty and purified mutant proteins were analyzed on 12.5% SDS-PAGE followed by electrophoretic transfer to the nitrocellulose membrane and staining with Ponceau S stain. Notice that because Ponceau S is a general protein stain, all proteins that are present in detectable ambound show up on this image. It appears that the purified mutants are free of major contaminating proteins.
Western Blot of Mutant CheY Another gel with sampls identical to the one shown above was transferred to another nitrocellulose membrane using the dual-slot Bio-RAD Mini Trans-Blot Electrophoretic Transfer Cell. After transfer of the separated proteins, the membrane was blocked with gelatin and probed with Ni-NTA-HRP conjugate overnight. The location of bound CheY was revealed via formation of colored insoluble precipitate in horseradish peroxidase-catalyzed reaction between 4-chloro-1-naphtol and hydrogen peroxide. Notice that because Ni-NTA binds specifically to to His-tagged protein, only CheY shows up in this image.
Mass Spectrometry of CheY The identity of the triple mutant CheY was analyzed by electrospray time-of-flight mass spectrometry. Purified protein was dialyzed extensively against deionized water and 20 µmolar sample was mixed with an equal volume of acetonitrile to facilitate evaporation of the solvent. The protein was ionized by addition of formic acid to a final concetration of 0.5 %. The image on the right shows the observed mass spectrum with a characteristic charge envelope arising from positively charged ions of intact CheY. Each major peak differs from the previous one by one charge unit. Analysis of this spectrum yields molecular mass of 15053 for the neutral protein molecule. This value is in a good agreement with the expected value for the triple mutant of CheY.
Binding of FliM: Fluorescence Study The biological function of CheY in bacterial chemotaxis is due to its ability to temporarily interact with the motor protein FliM. Participants in the course characterized the binding of the FliM peptide to the purified CheY protein using fluorescence spectroscopy. The binding of FliM will lead to significant quenching of the intrinsic fluorescence of CheY because the environment around the emitting tryptophan residue changes significantly upon binding. The extent of fluorescence quenching is proportional to the fraction of bound CheY. Analysis of fluorescence quenching as function of FliM concentration allows determination of the dissociation constant for the CheY-FliM complex. The image on the right shows results from one such experiment, yielding Kd value of about 0.7 µM.
NMR Characterization of FliM binding The binding of FliM to the purified triple mutant of CheY was studied by nuclear magnetic resonance spectrometry. NMR allows to monitor which CheY residues are involved in binding by comparing chemical shifts of protons in the free and bound forms of the protein. The image on the right shows the most upfield region of the proton spectrum. The methyl group of residue Val 95 that is involved in the binding of FliM appears at about 0.09 ppm as a sharp (ca 15 Hz) peak in the absence of FliM peptide. This peak shifts to -0.44 ppm in the FliM bound form and becomes broader (ca 38 Hz), indicating that the half-life of the complex in 25 mM Na-phosphate is well over one millisecond.
Crystallization of CheY Crystallization trials were set up to crystallize CheY and lysozyme (as a control). Protein crystallization is a required step for the determination of high-resolution structures by X-ray crystallography. Solutions of purified CheY were mixed with different precipitating agents in the hanging drop method. After two days, drops were examined under the polarizing microscope and few conditions that gave rise to protein crystals were identified. The image on the right shows a large lysozyme crystal while a smaller CheY crystal can be seen here here.
Participant Dinner and Discussion After successful three weeks, the course participants and instructors met in Palazzio Trattoria to enjoy some food and drinks, no LB broth this time!

Useful science links

Pymol Examples here at UCSB *with scripts*
Electronic Journals at UCSB
Google Scholar Web Search Engine
Biology Workbench
ExPASy Proteomics Server
Protein Data Bank
Periodic Table of the Elements
Cell and Molecular Biology Protocols Online

UCSB links

UCSB General Catalog
UCSB Campus Map
UCSB Gold Login
UCSB Umail Access
UCSB Environmental Health and Safety