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We have introduced a course named ''modern tech- niques in protein ... lar courses. ...... biology to undergraduate biology students—An illustration of protein.
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BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 36, No. 1, pp. 43–54, 2008

Laboratory Exercises Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization* Received for publication, March 11, 2007, and in revised form, July 22, 2007 Yifeng Wu†, Yangbin Zhou†, Jiaping Song, Xiaojian Hu, Yu Ding‡, and Zhihong Zhang From the Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China

We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or pT7His (His10-mDsRed) prokaryotic expression vectors. Then the fluorescent proteins were expressed in Rosetta (DE3) pLysS by IPTG induction or autoinduction. We purified the fluorescent proteins by affinity chromatography (Amylose and metal ionchelating column), anion-exchange chromatography (High Q column), size exclusive chromatography (Sephacryl S-200 column), and hydrophobic interaction chromatography (Methyl HIC column) to exhibit the protein-purification techniques. After purification, the fusion protein MBP-EGFP was cleaved by TEV protease. The recombinant mDsRed protein was crystallized by hanging drop vapor diffusion technique to show students the basic operation of crystallization. The whole procedure can be monitored real time by naked eyes when using fluorescent proteins. The demonstration of expression, purification, crystallization, and protease cleavage became much more vivid and interesting, which greatly deepened the students’ understanding of modern protein-science techniques. Keywords: Green fluorescent protein, red fluorescent protein DsRed monomer, chromatography purification, protein crystallization, TEV protease cleavage. processes of many biological activities within the cell, including protein folding, and transport. The GFP gene can also be maintained through breeding, and it was used to develop versatile reporter systems for genetic analysis. Different mutants of GFP have been engineered over the last few years. The enhanced green fluorescent protein (EGFP)1 was one of the most famous mutants with increased stability and fluorescence efficiency. EGFP shifts its major excitation peak from 395 to 488 nm while the peak emission remains at 507 nm [3]. Using commercial vectors such as pEGFP-N1/pEGFP-C1, target genes can be easily located in vivo with an N-terminus or C-terminus EGFP tag. Fluorescence resonance energy transfer (FRET), a common technique used to study protein–protein interaction, requires at least two kinds of fluorescent proteins with different excitation and emission spectrum. Besides the most popular FRET pair: cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), GFP and RFP pair is also widely used [4].

We have introduced a course named ‘‘modern techniques in protein structural and functional analysis’’ to the senior undergraduate students or the first-year graduate students who studied biology but had not yet taken similar courses. The course focused on the whole process of cloning, expression, purification, functional characterization and structural analysis of a specific protein. Green and red fluorescent proteins (GFP and RFP) were used in the laboratory sections, which greatly increased the students’ interests and minimized the specialized equipment usage at the same time. The GFP, a small protein comprised 238 amino acids from the jellyfish Aequorea victoria, emits green fluorescent light when exposed to blue light [1]. It is stable under most physiological environment as a weak dimer [2]. Nowadays, GFP has already become an excellent marker for gene expression and protein localization in cellular and molecular biology. It is also used to demonstrate the dynamic

* The work was supported by the grant 30500113, 30600107 and 30670499 from the National Natural Science Foundation of China. Y.B. Zhou was supported by the National Talent Training Fund in Basic Research of China (No. J0630643) and Science Innovation Funds from Fudan University. ‡ To whom correspondence should be addressed. Fax: þ8621-65650149. E-mail: [email protected]

1 The abbreviations used are: EGFP, enhanced green fluorescent protein; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; BCA, bicinchoninic acid; IPTG, isopropyl b-D-thiogalactopyranoside; mDsRed, DsRed monomer; Ni-NTA, nickel–nitrilotriacetic acid; RFP, red fluorescent protein; TEV, tobacco etch virus.

† These authors contributed equally to this work. This paper is available on line at http://www.bambed.org

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DOI 10.1002/bmb.117

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BAMBED, Vol. 36, No. 1, pp. 43–54, 2008

TABLE I Sequences of synthesized oligonucleotide primers used in cloning the EGFP gene into vector pMAL-c2x and mDsRed gene into pT7His Primer

Sequencea

N1 N2 C1 N3 C2

50 -ACTGAATTCGGCGGAGGCTCAGAAAACCTGTATTTTCAGGGCGGATCA-30 50 -TTTCAGGGCGGATCAGGCTCAGGAGGCGGATCCATGGTGAGCAAGGGCGAG-30 50 -TGCCTGCAGCTCGAGTTACTTGTACAGCTCGTC-30 50 -GTCGGATCCGAAAACCTGTATTTTCAGGGAATGGACAACACCGAGGAC-30 50 -GCGCTCGAGCTACTGGGAGCCGGA-30

a N2 and C1 were used in the first round of nested PCR. N1 and C1 were used in the second round of nested PCR to clone EGFP. N3 and C2 were used to clone mDsRed. The sequences cut by respective restriction endonucleases (N1: EcoR I, C1: Pst I, N3: BamH I, C2: Xho I) are underlined. The TEV recognition sequence in N1, N2, and N3 are italized.

DsRed, a RFP from Discosoma coral, was first reported in 1999 and soon became popular in biotechnology applications [5]. As a distinct label for multicolor tracking of fusion proteins in vivo, DsRed extended the spectrum of available colors from cyan-yellow (CFP and YFP) to red. However, the original DsRed protein was a tetramer, which hindered its application as a fusion tag. Several approaches had been tried to mutate the DsRed tetramer to a monomer [6, 7]. One successful ‘‘DsRed monomer’’ (mDsRed) was commercialized by Clontech. Besides a good FRET pair with GFP, mDsRed is also widely used in flow cytometry and fluorescence microscopy. Although many improvements have been made in X-raybased structural biology, the most challenging step is still the crystallization step. Our curriculum paid much attention to these techniques. The GFP and RFP (MBP-EGFP and His10-mDsRed) can be easily prepared by prokaryotic expression system and are visible to the naked eyes; therefore, the expression and purification processes can be observed without traditional UV spectroscopicmeter. This practice increased the success rate and caught the students’ attention. Several educators have published their attempts to use GFP in their courses [8–11]. Here, we introduced both RFP and GFP to expand the applications of fluorescent proteins in education, described the whole procedure to demonstrate the cloning, expression, purification, crystallization, and protease cleavage, and finally discussed several specific problems that the students may face in the educational practice. EXPERIMENTAL PROCEDURES

Equipments The equipments used are as follows: automation PCR (Eppendorf Mastercycler Gradient PCR), orbital constant temperature shaking incubator (16–37 8C), water bath (16–95 8C), low-pressure chromatography system (Bio-Rad’s LP System), electrophoresis system (Bio-Rad’s Mini-PROTEAN 3 system), constant temperature incubator (18 and 37 8C), and fluorescence microscope (Olympus BH-2).

Materials The vector pEGFP-N1 and pDsRed-Monomer-N1 were purchased from Clontech (Palo Alto, USA). The bacterial (Escherichia coli) hosts DH5a, Rosetta (DE3) pLysS, the vectors pET21a, and the protein markers were obtained from Novagen (Madison, WI). KOD plus Pfu polymerase and DNA ligation kit were purchased from Toyobo (Osaka, Japan). Nucleotides, agarose gel, DNA extraction kit, and PCR purification kit were purchased from Roche Diagnostics (Indianapolis, IN). Primers synthesis and DNA sequence analysis were performed by Invitrogen (Shanghai, China). Nickel-nitrilotriacetic acid (Ni-NTA)

Superflow column matrix was obtained from QIAGEN (Chatsworth, CA). Sephacryl S-200 HR and Phenyl Sepharose 6 FF matrix were purchased from Amersham Biosciences (Piscataway, NJ). High Q and Methyl HIC Cartridges were from BioRad (Hercules, CA). The restriction endonucleases, vector pMAL-c2x, and amylose resin were from New England Biolabs (Ipswich, MA). Bicinchoninic acid (BCA) protein assay reagent kit was from Pierce (Rockford, IL). b-Mercaptoethanol, EDTA, HEPES, imidazole, isopropyl b-D-thiogalactopyranoside (IPTG), D-glucose, D-lactose, D-maltose, and Triton X-100 were from Sigma (St. Louis, MO). PEG 8000 and other reagents for crystallization were from Fluka (Buchs, Switzerland). Amicon Ultra15 centrifugal filter (MWCO 10,000) was obtained from Millipore (Bedford, MA). All other reagents were of analytical grade.

Cloning the EGFP and mDsRed coding region into pMAL and pT7His vector We have previously reconstructed an expression vector named pT7His-containing N-terminus His10 and C-terminus His6 tag from vector pET21a; the detailed vector construction procedure was similar to that of pT7470, which contains N-terminus His6 and C-terminus His6 tag [12]. We amplified the CDS of EGFP and mDsRed using pEGFP-N1 and pDsRed-Monomer-N1 as the templates, respectively. To improve the efficiency of tobacco etch virus (TEV) protease cleavage, we amplified the EGFP CDS for two rounds to add a flexible linker sequence (the primers used were listed in Table I). The PCR reaction products were double digested by EcoR I and Pst I (EGFP), and BamH I and Xho I (mDsRed), respectively, and then ligated to the double-digested expression vector pMAL-c2x or pT7His (Figs. 1A and 1B). The ligation mixture was finally transformed to E. coli host DH5a. The positive pMAL-EGFP and pT7His-mDsRed clones were selected and sequenced for verification. Table II summarizes the whole timetable of the curriculum. The cloning process took 3 weeks (weeks 1–3).

Expression of pMAL-EGFP and pT7His-mDsRed The verified pMAL-EGFP and pT7His-mDsRed plasmids were transformed into E. coli strain Rosetta (DE3) pLysS for protein expression. After the colony bacteria grew overnight at 37 8C in 5-mL LB medium with 100 lg/mL ampicillin, 0.1 mL bacterial suspension was then transferred into 5-mL fresh LB medium and grew in 37 8C shaker (300 rpm) to OD (600 nm) of 0.6. The cells were induced by adding IPTG. We tested different concentrations of IPTG from 0.01–5 mM IPTG, various temperatures from 16 to 37 8C, and induction time from 1 to 8 hours. At the same time, large quantity of recombinant MBP-EGFP and His10-mDsRed was prepared by autoinduction [13] as follows. The cells were first cultured in 250-mL autoinduction medium at 37 8C. After the OD (600 nm) reached 0.6 (around 4 hours), the cells were cooled down to 25 8C and shaken at 300 rpm overnight. The cells were collected by centrifugation at 6,000 3 g for 15 minutes and preserved at 220 8C. The expression process took 1 week (week 4).

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FIG. 1. Cloning and expressing of green and red fluorescent protein. A: Overall cloning scheme to produce pMAL-EGFP construct. All plasmid maps were created by the Vector NTI software. The MBP-EGFP expression vector pMAL-EGFP was created by ligation of two parts: the linearized pMAL-c2x and the digested EGFP PCR amplification product. The complementary sticky ends created by the two restriction enzymes (EcoR I and Pst I) ensured proper directional ligation of PCR product with the vector. The translation of the resulting MBP-EGFP was under the control of the inducible tac promoter. B: Overall cloning scheme to produce pT7His-mDsRed. The His10-mDsRed expression vector pT7His-mDsRed was created by ligation between the linearized pT7His and the digested mDsRed PCR amplification product. The translation of the resulting His10-mDsRed was under the control of the inducible T7lac promoter. C: Small scale expression of MBP-EGFP and His10-mDsRed. The bacteria were cultured in 5-mL test tube and induced by 1 mM IPTG at 25 8C overnight. 1) Uninduced MBP-EGFP bacteria in culture medium; 2) induced MBP-EGFP bacteria in culture medium; 3) amylose affinity-purified MBP-EGFP; 4) uninduced His10-mDsRed bacteria in culture medium; 5) induced His10-mDsRed bacteria in culture medium; 6) Ni-NTA affinity-purified His10-mDsRed. D: Large scale expression of MBP-EGFP and His10-mDsRed in 250-mL culture medium. The left photo shows MBP-EGFP, His10-mDsRed, and another nonfluorescent control protein in the 2-L flask after 25 8C overnight autoinduction. The right photo shows the collected 250 mL induced MBP-EGFP and His10-mDsRed bacteria in the 300-mL centrifuge tube.

Week 5

Affinity chromatography

Week 6

Week 4

Purify MBP-EGFP by Amylose column

Verify the positive clones Plasmid purification by colony PCR, double digestion and sequencing; transform the positive plasmid to Rosetta strain for expression Incubate the transformed IPTG induction; E. coli Rosetta strain; autoinduction IPTG induction of pMALEGFP and pT7HismDsRed for expression test; autoinduction of pMAL-EGFP and pT7His-mDsRed for large-scale production Purify His10-mCherry by Ultrasonic lyzing cells; NiNi-NTA column NTA column chromatography Amylose column chromatography

Ligation; competent cell preparation; transformation

Week 3

Week 2

PCR; DNA electrophoresis; DNA gel extraction

Amplify the CDS of EGFP and mDsRed by PCR; double digest and purify the DNA fragments of EGFP and mDsRed as well as the prepared plasmids Ligate the digested EGFP and mDsRed fragments with the vectors; transform to E. coli DH5a strain for cloning

Content

Week 1

Time

Protein expression

Molecular cloning

Topic

Specific technology to learn

Low DNA recovery from agarose gel

Failure of PCR

Problems encountered by the students

Basic protein purification strategy; principle of affinity chromatography

Introduction of prokaryocyte expression system; principle of the autoinduction system

The history and principle of DNA sequencing technique

Part of the target protein flowed through the amylose column

Low yield of the target protein

Basic information about the Low efficiency of vectors used in this transformation course; difference between the cloning and expression E. coli strains

The history, structure, and biological applications of the fluorescent proteins

Background and introduction

TABLE II Timetable of the one semester course ‘‘Modern techniques in protein structural and functional analysis’’

Normal, the binding ability of amylose column is not very strong

Add detergents and extend the ultrasonic lyzing time

Make control group to examine the efficiency of the competent cells. If it is low, prepare fresh competent cells and store at 270 8C; if it is normal, try to examine the activity of the DNA ligase and perform all the experiment carefully

Try gradient PCR to determine the best annealing temperature Elevate the temperature of DNA elution buffer

Suggestions

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Week 10

Week 11

Week 12

Size exclusive chromatography

Hydrophobic interaction chromatography

Crystallization

Week 13

Week 9

Week 8

Week 7

Time

Anion exchange chromatography

Protease cleavage

Topic

Separate the mixed MBPEGFP and His10mDsRed by Sephacryl S-200 column Separate the mixed MBPEGFP and His10mDsRed by Methyl HIC column Ultrafiltration mDsRed to change the buffer; determine the protein concentration, dilute it to the proper value; crystallize mDsRed by hanging drop diffusion method Observe the crystal; open experiments and discussion

Dialyze the cleaved mDsRed; purify mDsRed by High Q column

Crystal observation by microscope

Ultrafiltration; hanging drop diffusion method

Hydrophobic interaction chromatography

Size exclusive chromatography

Dialysis; anion exchange chromatography

BCA method; protease Determine the protein cleavage concentration of MBPEGFP and His10mDsRed by BCA method; cleave MBPEGFP and His10mDsRed by TEVSH protease Examine the efficiency of SDS-PAGE; BandScan the cleavage by SDSsoftware PAGE; analyze the gel by 1D gel image software

Content

Specific technology to learn

TABLE II (Continued)

Target protein cannot bind to the column

Low SDS-PAGE gel quality

Low cleavage efficiency of TEVSH protease

Problems encountered by the students

Review of the whole semester course and discussion

Principle of hydrophobic interaction chromatography; choice of HIC media Introduction of crystallization methods and the X-ray-based structure resolving work

Normalize the samples for loading Examine the conductance of the solution, dialyze longer if the conductance is too high Concentrate the loading sample by ultrafiltration to a small volume Try changing the buffer system

Use fresh prepared TEVSH protease, dispense into small part and store at 220 8C

Suggestions

Mixing the protein and Work more precisely; diffusion buffer may have add 1-2 more drops deviation per well to balance the deviation Low crystal quality or no Concentrate the crystal proteins; crystallize a standard sample prepared by teacher

Principle and application of Low resolution size exclusive chromatography

Principle and application of Low separative efficiency anion exchange due to large volume of chromatography loading sample

Principle of SDS-PAGE

Methods to determine protein concentration; protease cleavage principle and application

Background and introduction

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BAMBED, Vol. 36, No. 1, pp. 43–54, 2008 Affinity purification of MBP-EGFP and His10-mDsRed

The collected cells from 250-mL autoinduction medium were thawed and resuspended in 100-mL buffer A (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 10% glycerol). The cells were lysed by sonication. After centrifugation at 20,000 3 g for 20 minutes, the MBP-EGFP supernatant was loaded on a 5-mL (7 3 1 cm) Amylose column, and His10-mDsRed supernatant was loaded on a 5-mL (7 3 1 cm) Ni-NTA Superflow column. The Amylose column was washed by 20-mL buffer A-containing 500 mM NaCl and then eluted by amylose elution buffer (10 mM D-maltose in buffer A). The Ni-NTA column was washed by 20-mL buffer A-containing 40 mM imidazole and then eluted by Ni-NTA elution buffer (500 mM imidazole in buffer A). The whole process was analyzed by SDS-PAGE. Each SDS-PAGE sample was divided into two fractions: one fraction was treated in 95 8C water bath for 5 minutes while the other was not. The heat-denatured and nonheated fractions were separated on 12% SDS-PAGE gels, respectively. The gel of non-heated denatured fraction was excited by 365-nm UV light and recorded by the gel-imaging system. The gel of heat denatured fraction was stained by Coomassie Brilliant blue R-250, scanned, and analyzed by BandScan Software Version 4.30 (Glyko). The affinity purification took 2 weeks (weeks 5 and 6).

Cleavage of MBP-EGFP and His10-mDsRed by TEV protease The plasmid pTH24-TEVSH encoding an improved solubility TEV protease was a gift provided by Dr. Helena Berglund, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Sweden. TEVSH protease was expressed and purified following Berglund’s protocol [14]. The concentration of MBPEGFP, His10-mDsRed, and TEVSH protease was determined by BCA method according to the protocol of BCA protein assay reagent kit (Pierce). The recombinant MBP-EGFP protein purified by Amylase column was diluted to 1 mg/mL. Five microliters (1 mg/mL) TEVSH were directly added to 1 mL MBP-EGFP to start the protease-cleavage reaction in 16 8C water bath; 50-lL reaction buffer was taken out at 2, 5, 10, 20, 30, 60, 90, 120, 180, 240, 300, and 600 minutes, respectively. The samples were immediately mixed with 25 lL 33 SDS-PAGE loading buffer to stop the reaction after they were taken out. These samples were analyzed by 12% SDS-PAGE gel. In another experiment, TEVSH (100 lL; 1 mg/mL) was added to the imidazole-eluted His10-mDsRed (around 10 mL; 5 mg/ mL); the reaction took a week at 4 8C; then the solution was dialyzed against anion-exchange buffer A (50 mM Tris–HCl, pH 8.0, 10% glycerol) twice for 2 hours each. The TEV protease-cleavage process took 2 weeks (weeks 7 and 8).

Anion-exchange purification of mDsRed The dialyzed mDsRed (contains TEVSH) solution was loaded on a 5 mL (7 3 1 cm) High Q column pre-equilibrated with anionexchange buffer A. The proteins were eluted with a linear 0–0.5 M NaCl gradient by mixing anion-exchange buffer A and anionexchange buffer B (50 mM Tris–HCl, pH 8.0, 1 M NaCl, 10% glycerol) automatically. Samples were analyzed by SDS-PAGE. The anion-exchange purification lesson took 1 week (week 9).

Size exclusive and hydrophobic interaction chromatography to separate MBP-EGFP and His10-mDsRed MBP-EGFP (around 5 mg/mL) was mixed with His10-mDsRed (around 5 mg/mL) at ratio of 1:1; 0.2-mL mixture was loaded on a 10 mL (14 3 1 cm) Sephacryl S-200 column, which was equilibrated with PBS. The collected fractions were analyzed by 12% SDS-PAGE.

One milliliter mixture of MBP-EGFP and His10-mDsRed was loaded on a 5 mL (7 3 1 cm) methyl HIC column pre-equilibrated with HIC buffer (buffer A contains 1 M (NH4)2SO4). The flow-through and the eluate of 1–0 M (NH4)2SO4 gradient were collected and analyzed by SDS-PAGE. The size exclusive and hydrophobic interaction chromatography lesson took 2 weeks (weeks 10 and 11).

Crystallization of mDsRed The buffer of the purified mDsRed protein was changed to crystallization buffer A (50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM b-mercaptoethanol) by ultrafiltration. Then, the mDsRed protein was further concentrated to about 20 mg/mL by ultrafiltration. The concentration of mDsRed was determined by BCA method. Crystallization was carried out by hanging drop diffusion method at 18 8C. Concentrated mDsRed was diluted to 9 mg/mL or 6 mg/mL by crystallization buffer A; then the 1 lL diluted mDsRed was mixed with 1 lL reservoir buffer. Differently shaped crystals appeared overnight under the conditions of A–D, respectively. Conditions are listed as follows: well A–C (9 mg/mL mDsRed)—A, 15% PEG 8000/0.1 M Na Ac anhydrous pH 4.6; B, 18% PEG 8000/0.1 M Na Ac anhydrous pH 4.8; C, 25% PEG 8000/0.1 M Na Ac anhydrous pH 5.8. Well D (6 mg/mL mDsRed): 20% PEG 8000/0.1 M Na Ac anhydrous pH 5.0. The crystals were observed under fluorescence microscopy (white light and green light excitation, respectively). The crystallization lesson took 2 weeks (weeks 12 and 13). RESULTS AND DISCUSSION

Cloning and expression of pMAL-EGFP and pT7His-mDsRed Figs. 1A and 1B show the overall cloning scheme of producing the pMAL-EGFP and pT7His-mDsRed, respectively. To remove the MBP tag or His10 tag, TEV protease-recognition sequence (Glu-Asn-Leu-Tyr-Phe-Gln;Gly or ENLYFQ;G; ; shows the cleavage site) was incorporated into the N terminus of the forward primer. The positive clones were verified by colony PCR and doubledigestion test in class, and finally sequencing. Then, the students were instructed to align the sequence with a reference sequence. Correct pMAL-EGFP and pT7HismDsRed constructs were then transformed into Rosetta (DE3) pLysS for expression experiment. In the molecular-cloning section, most students got the positive clones in the end, though the students still faced several problems including failure in PCR, low DNA recovery from agarose gel, low quality of ligation reaction, and low efficiency of transformation (listed in Table II). Carefully handling the DNA sample can solve more than 80% of the problems. The rest problems may be solved by trying experiments listed in the ‘‘Suggestions’’ column in Table II. We performed two rounds of PCR using primers N1 and N2 to add linker sequences in the N terminus of EGFP gene. Previously, we have tried ordinary PCR and got positive clones. But when we performed TEVSH proteasecleavage experiment using these clones (discuss later), the efficiency of the cleavage was much lower than expected (data not shown). This phenomenon probably resulted from the steric hindrance of the TEV-cleavage site partially buried by MBP and EGFP. So, we redesigned the primers that contain sequences encoding linker peptides

49 before (GGGS) and after (GSGSGGGS) the TEV-cleavage sequence (ENLYFQG) as shown in Table I. The redesigned MBP-EGFP protein with linkers could be efficiently cleaved by TEVSH. We also added a BamH I site after the TEV-recognition site to allow other interesting genes to be conveniently inserted into this expression vector. In pT7His-mCherry primer design, we placed the TEVrecognition sequence after the BamH I site instead of that we discussed previously. The reason is as follows: as the mDsRed would be used in the demonstration of protein crystallization, additional residues may increase the flexibility of target protein, which might undermine crystal precipitation. In our design, after being cleaved by TEVSH, the recombinant protein has only one additional glycine at N terminus. Then the putative flexibility problem would not bother anymore. In the previous two steps of primer design, students were inspired to think critically about arranging doubledigestion sites according to protein structure and steric effects at the same time. In the theoretical sections, students were learning the basic principles of pT7His and pMAL expression system, the host strain Rosetta (DE3) pLysS characteristics, the strong promoter tac (pMAL) and T7lac (pT7His) usage, the MBP and poly-histidine tag property, and the induction principles. In the laboratory sections, the induction time, temperature, and IPTG concentration were tested. Students could detect the effect of these factors directly by observing the color changes with naked eyes. The protein synthesis reached a constant level around IPTG concentration of 0.5 mM and induction time of 6 hours (data not shown). Although the cultured bacteria grew much slower at 25 8C or even lower temperature, the students found that the fluorescent was stronger than 37 8C (data not shown). This shows that the lower temperature can slow down protein synthesis rate to avoid the formation of inclusion body. If the folding machinery in E. coli is saturated by fast synthesis, the recombinant proteins are easy to aggregate. Besides, lower temperature also decreased the protease activity. Figs. 1C and 1D show the small and large scale expression of MBP-EGFP and His10-mDsRed in bacteria; the difference before and after induction can be easily seen. Expression of target protein by autoinduction, a method developed by F. William Studier, is becoming more and more popular [13]. Some researchers have previously found that sporadic, unintended induction of expression resulted from small amounts of lactose in culture media; but the presence of glucose would prevent the induction by lactose. The cultured cells first use glucose but not lactose in low density; when they grow to high density and the glucose is depleted, the induction by lactose will automatically start. Autoinduction allows an efficient screening of many clones in parallel for expression and solubility, since the students need not wait until the OD (600 nm) reach 0.6 to add IPTG or synchronize all the cells to induction. The only thing they need to do is to harvest the cells next morning. The autoinduction system can also increase the yields of target proteins; typically, we can get more than 600-mg MBP-EGFP or His10-mDsRed from 1 L autoinduction culture medium; whereas, in contrast, the

IPTG induction produces around 100-mg target protein. Another advantage of autoinduction is that the medium is much cheaper than IPTG. From Figs. 1C and 1D, we found that the naked eyes are more sensitive to the red color than the green, especially when the background is blue. Therefore, we linked mDsRed but not EGFP to the His10 tag, which would be further purified by blue Ni-NTA resin.

Affinity purification of MBP-EGFP and His10-mDsRed The induced cells were lysed by sonication followed by centrifugation. After centrifugation, students would find that most of the green/red color appears in the soluble fraction, and the insoluble debris are just greenish or reddish, showing that the properly folded target proteins are mainly in the supernatant. Fluorescent proteins were very stable. The MBP-EGFP and His10-mDsRed in the sonication supernatant were stable at room temperature for at least 1 week; the purified MBP-EGFP or His10-mDsRed were stable at room temperature for at least half a year (data not shown). So, in the whole semester’s experiment, we did not need to add any protein stabilizer and protease inhibiter (PMSF, etc.), which are extremely toxic to the eyes, skin, and the mucous membranes of the respiratory tract. When the MBP-EGFP was loaded onto a Amylose column, the color of the column quickly shifted from white to green from the upper side; whereas, the flow-through fraction was colorless (if the purification was run on a chromatography system, the OD of 280 nm is still high, there were none-binding proteins that flow through the column). After the whole column became green, the flow-through fraction also turned green, showing that the column was saturated with MBP-EGFP protein. Typically, one fourth of the MBPEGFP from 250-mL culture would saturate a 5-mL Amylose column. This experiment could be repeated several times to purify all the harvested proteins. His tag was used much more widely than MBP tag, and so immobilized metal affinity chromatography (IMAC) purification was also more widely applied than Amylose purification. Fig. 2A shows the visualization process of purification of red His10-mDsRed on a blue Ni-NTA column under natural light. The whole process was also monitored by a low pressure chromatography system (Fig. 2B, upper); the collected numbered eluate was shown respectively (Fig. 2B, lower). The expression and purification process was also analyzed by SDS-PAGE. Fig. 2C shows that the MBP-EGFP or His10-mDsRed were present before the induction as very faint bands and were substantially enhanced after induction. Most target proteins bound to the column, and the flow-through fraction did not contain obvious target proteins. The eluted affinity purification fraction contained >90% purity target proteins. Fig. 2D shows the result of fluorescence pattern emitted by EGFP or mDsRed protein after SDS-PAGE electrophoresis. EGFP and mDsRed were stable under most harsh conditions. Even in the denatured SDS-PAGE system, EGFP and mDsRed still kept their fluorescence. In the SDS-PAGE gel, the active fluorescent pro-

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FIG. 2. Purification of MBP-EGFP and His10-mDsRed by affinity chromatography and ion-exchange chromatography. A: Visualization of His10-mDsRed purification procedure under natural light. The photo shows, from left to right, the color variation of a Ni-NTA column before loading, start loading, after loading, start imidazole elution, and almost all His10-mDsRed were eluted, respectively. B: The curves of absorbance280nm (blue) and conductivity (red) versus time for His10-mDsRed purified by Ni-NTA column in a low pressure chromatography system. The supernatant was loaded (0–6 minutes), washed by buffer A-containing 20 mM imidazole (6–17 minutes) and 40 mM imidazole (17–33 minutes), and then eluted in a 20–500 mM imidazole gradient (33–58 minutes). The lower photo shows the His10-mDsRed loading cell supernatant (first tube), Ni-NTA column flow-through fraction (second tube), and No. 2–17 fraction eluate according to the upper picture. C: SDS-PAGE analysis of the expression and purification of MBP-EGFP and His10-mDsRed. Marker, Novagen’s Perfect Protein markers; (2), uninduced bacterial lysate; (þ):, supernatant of induced bacterial lysate; F.T., flow through of affinity column (MBP-EGFP: Amylose column, His10-mDsRed: Ni-NTA column); (E) 10 mM maltose-eluted MBP-EGFP; (E1) 500 mM imidazole-eluted His10-mDsRed; E2, 0.15 M NaCl-eluted mDsRed (after His10 tag cleaved by TEVSH). D: Fluorescence image of the expression and purification of MBP-EGFP and His10-mDsRed. The samples loaded were the same as (C) without denaturing by heat. The picture was captured in an ordinary gel-imaging system (fluorescence was excited by 365-nm UV light). E: The curve of absorbance280nm (blue) and conductivity (red) versus time for mDsRed purified by High Q column in a low pressure chromatography system. The sample of mDsRed/His10 tag/TEVSH mixture (after His10 tag cleaved by TEVSH) was dialyzed against anion changer buffer A (50 mM Tris–HCl, pH 8.0, 10% glycerol). Then the sample was loaded on the High Q column (22–30 minutes), washed by anion changer buffer A (30–40 minutes), and eluted in a 0–500 mM NaCl gradient. The column was cleaned up by 1 M NaCl (40–65 minutes) (fractions 6–9 were mixed as ‘‘E2,’’ analyzed by SDS-PAGE).

tein appears as a thinner band with an apparently larger molecular weight when compared with the denatured fluorescent protein. The thinner band might indicate that the fluorescent protein has a consistent and tight barrel structure in this condition. The band with an apparently larger molecular weight may be the result that SDS cannot bind

the active fluorescent protein as much as other proteins. But the fluorescent proteins were not as heat stable as Taq polymerase; if they are denatured at 95 8C for 5 minutes, the fluorescence will disappear fully. We chose 365 nm UV to excite the fluorescent proteins because this experiment could be performed in an ordinary

51 DNA gel-imaging system. Almost every biological laboratory has such kind of apparatus. If a longer wavelength lamp was used as the excitation source, the emission strength of the fluorescent proteins would be much stronger. In this week’s course, the students’ interests gradually increased with beautiful colors emitted by EGFP and mDsRed. As a result, they became quickly acquainted with basic purification principles and characteristics of fluorescent proteins.

Cleavage of MBP-EGFP and His10-mDsRed by TEV protease Nowadays, it is common to express target protein with a specific tag and then to remove the tag after the affinity purification step by the action of a specific protease. These tags can increase the solubility and reduce proteolytic degradation of the target recombinant protein; they may also improve folding efficiency and simplify the purification and detection procedure. However, these tags may interfere with the structure and function of target protein, thus they are always removed by site-specific proteases. Several site-specific proteases are used, including thrombin, factor Xa, enterokinase, PreScission, and TEV protease. Within these proteases, TEV protease’s specificity and efficiency is highest, and the TEV protease can be easily produced by our own laboratory. We can produce more than 10-mg purified TEVSH (an improved solubility TEV protease kindly provided by Dr. Helena Berglund) from 250-mL autoinduction medium, and so we designed the TEVSH-cleavage experiment. Figs. 3A and 3B show the time course of MBP-EGFP cleaved by TEVSH protease at 16 8C. The reaction started immediately after adding TEVSH. After an overnight incubation, almost all the substrate MBP-EGFP was cleaved. The scanned gel was analyzed using BandScan Software Version 4.30 (Glyko); the quantity of uncleaved MBP-EGFP and cleaved MBP/EGFP was calculated. The TEVSH-cleavage completeness (%) was defined as follows: (quantity of cleaved MBPþEGFP)/(quantity of uncleaved MBP-EGFP þ cleaved MBPþEGFP). Fig. 3C shows the relationship between TEVSH-cleavage completeness (%) and time, in which the red line was the result of exponential decay fit using the Origin 6.0 software package (Microcal, Northampton, MA). Students were encouraged to analyze the SDS-PAGE gel by themselves.

Anion-exchange chromatography purification of mDsRed

FIG. 3. MBP-EGFP cleaved by TEVSH protease. A: SDSPAGE analysis of MBP-EGFP cleaved by TEVSH protease. Marker, Novagen’s Perfect Protein markers; TEVSH, the purified TEVSH used in the cleavage experiment; 0, 2, 5, 10, 20, 30, 60, 90, 120, 180, 240, 300, and 600, SDS-PAGE of protease-cleavage solution at time intervals (minutes) when the reaction was stopped. B: Fluorescence image of the MBP-EGFP cleaved by TEVSH protease. The samples loaded were the same as (A) but without heat denaturing. Fluorescence was excited by 365-nm UV light. C: The curve of completeness of the reaction versus cleaving time for the TEVSH protease cleaving MBP-EGFP experiment. The product (%) of the cleavage experiment was calculated according to the SDS-PAGE data analyzed using BandScan 4.30 Software. The figure was drawn by Origin 6.0 software package. The red line showed an exponential decay fit result.

Ion-exchange separation relies on reversible charge interactions between a charged biomolecule (such as a protein or nucleic acid) and an oppositely charged resinbased matrix. Because the pI of many proteins is below seven, anionic buffer system is commonly used, and anion exchange is more widely used than the cation-exchange chromatography. Commonly used strong ion-exchange medium includes quarternary ammonium (High Q, which is negatively charged) and methyl sulfonate (High S, which is positively charged). In this experiment, High Q was used to further purify TEVSH protease-cleaved mDsRed. In the pH 8.0-loading buffer, the cationic TEVSH protease and cleaved

His10 tag did not bind to the High Q column, while cleaved mDsRed bound to the matrix. The cleaved mDsRed was eluted at around 0.15 M NaCl. Other contaminants that eluted from Ni-NTA column could also be separated by the NaCl concentration gradient. Fig. 2E was a demonstration of mDsRed High Q purification. The right lanes in Figs. 2C and 2D show that the recombinant mDsRed protein became very pure after two steps of purification by Ni-NTA and High Q. Before recombinant technology and affinity purification came into widely use, ion exchange, hydrophobic inter-

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FIG. 4. Separation of MBP-EGFP and His10-mDsRed by Size exclusive chromatography. A: Visualization of separation of MBP-EGFP and His10-mDsRed mixture by a 14-cm Sephacryl S-200 column under natural light. The photo shows a series of photos taken after MBP-EGFP and His10-mDsRed mixture was loaded. B: The curve of absorbance280nm (blue) and conductivity (red) versus time for MBP-EGFP and His10-mDsRed mixture separating by Sephacryl S-200 column in a low pressure chromatography system. The column was equilibrated with PBS. The flow rate was 0.5 mL/minutes. The mixture of green and red fluorescent protein was loaded at 0.6–1 minutes (0.2 mL), 1 mL/fraction; fractions 3–10 (6–22 minutes) were collected and analyzed by SDSPAGE. C: The photo of separated protein solutions by Sephacryl S-200 size exclusive chromatography. Tube 1, amylose-purified MBP-EGFP; tube 2, Ni-NTA-purified His10-mDsRed; tube 3, loading fluorescent protein mixture (MBP-EGFP and His10-mDsRed); tubes 4–11, fractions 3–10 of Sephacryl S-200 size exclusive chromatography as in Fig. 4B. D: SDS-PAGE analysis of separation of MBP-EGFP and His10-mDsRed by Sephacryl S-200 size exclusive chromatography. Marker, Novagen’s Perfect Protein markers; MBP-EGFP, amylose-purified MBP-EGFP; His10-mDsRed, Ni-NTA-purified His10-mDsRed; mix, the mixture of MBP-EGFP and His10-mDsRed; E3-E10, fractions 3–10 of Sephacryl S-200 size exclusive chromatography as in Fig. 4B. E: Fluorescence image of the separation of MBP-EGFP and His10-mDsRed mixture by Sephacryl S-200 size exclusive chromatography. The samples loaded were the same as (D) but without heat denaturing.

action, and size exclusive chromatography were the most important protein-purification methods. Now, these methods are still important to high-quality protein purification. Traditional demonstrations always require a 280nm UV spectrometer to monitor the target proteins; but even with the spectrometer, students seem not to be interested in the tedious purification steps. However, using fluorescent proteins, they learned the basic working principle and arrangement of the automatic chromatography system quickly by watching the colored fluids moving in the transparent tubes. Also, they ruled out when and where the target protein bound to the matrix, and in which condition the protein moved and eluted. When the protein mixture of whole cell lysate was loaded on the column, the color would also point out the exact position where our target fluorescent protein was, thus gave more information than the UV absorbance, which only shows the absorbance of total proteins.

Separation of MBP-EGFP and His10-mDsRed by Size exclusive and hydrophobic interaction chromatography Size exclusive chromatography, also called gel filtration, separates biomolecules according to the differences between their sizes when they pass through a gel filtration medium packed in the column. Besides separate target protein, it is also widely used in rapid buffer exchange and desalting. To effectively teach the students about the principle, application, advantage, and disadvantage of size exclusive chromatography, we chose commonly used Sephacryl S-200 low pressure column and two fluorescent proteins, MBP-EGFP (MW 71.5 kDa) and His10-mDsRed (MW 27.9 kDa) of different molecular weight to demonstrate the purification process. Fig. 4A shows the time course of the separation by Sephacryl S-200 column under natural light: the color of the mixture of MBP-EGFP and His10-mDsRed was or-

53 ange; soon after the mixture enter the Sephacryl S-200 matrix, MBP-EGFP separated from His10-mDsRed; the green band ran faster than the red band. Fig. 4B shows the whole process recorded by a low-pressure chromatography system. The samples of purified MBP-EGFP, His10-mDsRed, their mixture, and fractions collected by size exclusive chromatography samples, respectively, were shown in Fig. 4C. Fig. 4D shows the SDS-PAGE result of all the samples in Fig. 4C. Fig. 4E shows their fluorescence pattern. Although in Fig. 4A, the separation of green and red bands was observed by naked eyes, Figs. 4B, 4D, and 4E show that the two peaks were partially merged, because the resolution of Sephacryl S-200 column was not high enough to separate 71.5 kDa (MBP-EGFP) and 27.9 kDa (His10-mDsRed) proteins completely. The students observed that the eluted target protein was significantly diluted during the size exclusive chromatography step. Now, they knew why the gel filtration step was always used as the last polishing step but not in the first capture step of protein purification. The students were also encouraged to measure several important values used in size exclusive chromatography, including void volume (Vo), total column volume (Vt), elution volume (Ve), and peak width (We) of MBP-EGFP and His10-mDsRed. Then, the resolution (Rs) was calculated by the equation: Rs ¼ 2(Ve2 2 Ve1)/(We1 þ We2). Although we performed this demonstration in a lowpressure chromatography system to record the UV absorbance, it can also be performed without a complicated chromatography machine. The gravity column also worked well (figure not shown). In addition, we also compared two hydrophobic interaction chromatography matrix in separating MBP-EGFP and His10-mDsRed. When the MBP-EGFP and His10mDsRed mixture-containing 1 M (NH4)2SO4 was loaded on a Phenyl Sepharose column, all the proteins bound to the matrix; His10-mDsRed can be washed out by 1–0 M (NH4)2SO4 gradient; but the MBP-EGFP’s-binding ability was so strong that even washed by the buffer without (NH4)2SO4, the protein still bound to Phenyl Sepharose. Then, we tested a weaker hydrophobic interaction matrix—Methyl HIC matrix. When the MBP-EGFP and His10-mDsRed mixture-containing 1 M (NH4)2SO4 was loaded, the His10-mDsRed direct flowed through, while the MBP-EGFP can be eluted by 1–0 M (NH4)2SO4 gradient. So, the hydrophobic interaction chromatography can be demonstrated by separating MBP-EGFP and His10-mDsRed mixture using Methyl HIC matrix. But, in this case, the resolution of Methyl HIC matrix was much lower than the High Q anion exchange (data not shown).

Crystallization of mDsRed Crystals of mDsRed were obtained under several crystallization conditions. Fig. 5 shows four typical mDsRed crystals with different shapes and sizes. By comparing the differences between the crystallization conditions, students were initiated to summarize the rules of mDsRed

FIG. 5. Crystallization of recombinant mDsRed protein in different conditions. All the crystals were observed with a fluorescent microscope (4 3 10) with white light (left) or excited by green light and filtered by a red filter (right). (A) 9 mg/mL mDsRed in 15% PEG 8000/0.1 M Na Ac anhydrous pH 4.6; (B) 9 mg/mL mDsRed in 18% PEG 8000/0.1 M Na Ac anhydrous pH 4.8; (C) 9 mg/mL mDsRed in 25% PEG 8000/0.1 M Na Ac anhydrous pH 5.8; (D) 6 mg/mL mDsRed in 20% PEG 8000/0.1 M Na Ac anhydrous pH 5.0.

54 crystallization. There were intriguing aspects in this week’s experiments. A lot of interesting questions were asked by the students. One question was whether the crystals were mDsRed protein or just salt crystals in the buffer. And the students verified the crystals by two methods: 1) the crystal was dissolved in an appropriate buffer and analyzed by SDS-PAGE; 2) the protein crystal was directly excited and observed it under a fluorescent microscope. In the crystallization conditions listed in Fig. 5, we only get needleshaped crystals, which is still not sufficient for X-ray diffraction. Adjusting the protein concentration or PEG concentration may increase the crystal quality. Besides the experiments discussed earlier, several students were also interested in the fluorescent proteins’ property and their application. The last section of this curriculum was an open experiment, in which the students were free to design experiments they were interested in. Several students tried to record the excitation and emission spectrum of GFP and RFP and to observe the shift of excitation and emission spectrum when changing the concentration, pH, ionic strength, or oxidative state. In conclusion, GFP and RFP have dramatically enhanced our laboratory curriculum quality; the students were effectively educated with a better understanding of the modern techniques of protein science, especially of several important initial steps of structural biology. With students’ naked eyes as the best monitor, the teaching of protein expression, purification, crystallization, and characterization would be no longer monotonous as before. Especially, all the four commonly used chromatography techniques (affinity, ion exchanger, hydrophobic interaction, and size exclusive chromatography) used in protein purification were demonstrated with higher inspirations. We are now expanding the application of GFP and RFP to other courses including biophysics, genetics, and molecular biology in our school.

BAMBED, Vol. 36, No. 1, pp. 43–54, 2008 Acknowledgment— We acknowledge Ruilin Zhang (Mayo Clinic, Rochester, Minnesota) for the critical review of the manuscript.

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