Chapter 7
A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins1
William H. Eschenfeldt, Lucy Stols, Cynthia Sanville Millard, Andrzej Joachimiak, and Mark I. Donnelly
Summary
Fifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate protein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors arc processed by the same protocols, which we describe here.
Key words: Structural genomics; High throughput; Protein purification; Ligation-independent cloning; Coexpression; In vivo proteolysis; Maltose-binding protein; TEV protease; TVMV protease
7.1. Introduction
A family of compatible ligation-independent cloning (LIC) vectors (I) was created to enable effective high-throughput cloning and purification of recombinant proteins for structural studies. All
'The submitted manuscript has been created by the University of Chicago as operator of Argonne National Laboratory under Contract No. W-31-109-ENG-38 with the U.S. Department of Energy. The U.S. government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the government.
Sharon A. Doyle (ed.), Methods in Molecular Biology: High Throughput Protein Expression and Purification, vol. 498 © 2009 Humana Press, a part of Springer Science + Business Media, Totowa, NJ Book doi: 10.1007/978-1-59745-196-3
105
106
Eschenfeldt et al.
the vectors contain a sequence encoding the tobacco etch virus (TEV) protease cleavage site (2) next to an Sspl site used for LIC (Fig. 7.1). The base vector, pMCSG7 (3), appends an N-termi-nal hexahistidine tag to proteins that is followed by the protease recognition sequence. Derivatives of the base vector (Table 7.1) add maltose-binding protein (4, 5), glutathione-S-transferase (6, 7), or a loop of GroES (8-10) to the leader, replace the his-tag with the S-tag (7, 11), incorporate a second protease cleavage site for in vivo tag removal (5, 12), or move the entire LIC region into different, compatible vectors to allow coexpression of proteins (5, 7). In all cases, expression is driven by T7 polymerase under control of the lac promoter in specific host strains (13). To introduce genes into the vector, LIC-compatible extensions are added through the use of specific primers during amplification by PCR (1). Following appropriate processing, the PCR product can be introduced into any member of the family by a standard LIC protocol. This chapter describes the manual, nonhigh-throughput LIC of genes into the pMCSG vectors. Chapter 8 describes plate-based methods for high-throughput applications.
The LIC process is identical for all members of the family (Fig. 7.2). The vector is first linearized by cleavage with Sspl then treated with T4 polymerase in the presence of dGTP only. Exo-nuclease activity of the polymerase hydrolyzes nucleotides from the 3' ends of the vector until it reaches a G residue, creating a 15-base, single-stranded 5' overhang. Conversely, treatment of appropriate PCR products with polymerase in the presence of only dCTP generates a complementary overhang. The two treated fragments are combined, allowed to anneal, and introduced into a suitable host. The host's native enzymes ligate then propagate the plasmid. In order to place nucleotides encoding a TEV cleavage site as close as possible to the introduced gene,
TEV site LIC site
lac operator T7 promoter \
± Insert Ssp His tag      \ /
T7 terminator
1
HmdlH
Nde I (ATG)
BamUl
Bglll
Kpnl
Fig. 7.1. Generalized organization of MCSG vectors. MCSG vectors encode an N-terminal leader sequence {anovt\ that terminates in an LIC region centered on an Sspl site. Restriction sites in and around the coding/cloning region allow insertion of protein or peptide modules into the leader (at Bgh\ and/or Kpni), replacement of the his-tag (A/cte/to Bgh\ or Kpn\), or transfer of the entire region to different backgrounds (AWel to BanM, HinD\\\ or Xhd). Derivatives (Table 7.1) add MBP, GST, or a loop of GroES (Sloop), replace the His-tag with the S-tag, and move these expression regions to pACY-CDuet-1 and pCDFDuet-1, allowing cotransformation with two or three vectors and coexpression of multiple proteins. Another modification, pMCSGI 9, positions untagged MBP followed by the TVMV protease recognition sequence (12) ahead of the His-tag, allowing in vivo removal of MBP by coexpressed TVMV protease.
A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins 107
Table 7.1
MSCG vectors for high-throughput ligation-independent cloning Parental
Vector	vector	Antibiotic	Leader sequence	MW (leader)3	kb	Purpose
PMCSG7	pET-21ab	Amp	N-His-TEV-LIC	2,755	5,286	production
pMCSG8	pMCSG7	Amp	N-His-Sloop-TEV-LIC	4,399	5,341	toxicity
pMCSG9	pMCSG7	Amp	N-His-MBP-TEV-LIC	43,713	6,147	solubility
pMCSGlO	pMCSG7	Amp	N-His-GST-TEV-LIC	29,046	5,961	solubility
pMCSGll	pACYC-Duet-lc	Cam	N-His-TEV-LIC	2,755	4,079	coexpression
pMCSG12	pACYC-Duet-1	Cam	N-His-Sloop-TEV-LIC	4,399	4,144	coexpression
pMCSG13	pACYC-Duet-1	Cam	N-His-MBP-TEV-LIC	43,713	4,940	coexpression
pMCSG14	pACYC-Duet-1	Cam	N-His-GST-TEV-LIC	29,046	4,754	coexpression
pMCSG17	pMCSG7	Amp	N-Stag-TEV-LIC	3,760	5,316	coexpression
pMCSG19	pMCSG7	Amp	N-MBP-TVMV-His-TEV-LIC	45,050/ 2,711d	6,441	production
pMCSG20	pMCSG17	Amp	N-Stag-GST-TEV-LIC	30,051	5,991	coexpression
pMCSG21	pCDFDuet-lc	Spec	N-His-TEV-LIC	2,755	3,852	coexpression
pMCSG22	pCDF-Duet-1	Spec	N-His-Sloop-TEV-LIC	4,399	3,906	coexpression
pMCSG23	pCDF-Duet-1	Spec	N-His-MBP-TEV-LIC	43,713	4,971	coexpression
pMCSG24 pCDF-Duet-1		Spec	N-His-GST-TEV-LIC	29,046	4,527	______________ coexpression
All 15 vectors are processed for LIC the same way and accept the same properly prepared PCR products. Abbreviations: Amp ampicillin; Cam chloramphenicol; Spec spectinomycin; His hexahistidine tag; TEV tobacco etch virus protease recognition sequence; LIC ligation-independent cloning site centered on an Sspl site; Sloop GroEL-binding loop of GroES; MBP maltose-binding protein; GST glutathiones-transferase; Staj; S-xag fragment of ribonuclease; TVMVtobacco vein mottling virus protease recognition sequence
■•Molecular weight removed by cleavage with TEV protease hVector pET-21a is a product of Novagen, Inc. (Madison, WI)
Vectors pACYCDuet-1 and pCDFDuet-1 are products of Novagen, Inc. (Madison, WI) dFirst value is molecular weight removed by cleavage by TEV protease without prior treatment with 1VMV protease. Second value is that removed after prior treatment with TVMV protease. TVMV protease removes 42,339 Da comprising untagged MBP and flanking amino acids
108      Eschenfeldt et al.
Vector
--CTGTACTTCCAATCCAAT - -GACATGAAGGTTAGGTTA
ATTGGAAGTGGATAACGG- -TAACCTTCACCTATTGCC- -
T4 polymerase dGTP
--CTG
--GACATGAAGGTTAGGTTA
ATTGGAAGTGGATAACGG--GCC--
PCR product
TACTTCCAATCCAATGCX----TAACATTGGAAGTGGATAA
ATGAAGGTTAGGTTACGY----ATTGTAACCTTCACCTATT
T4 polymerase dCTP
TACTTCCAATCCAATGCX----TAAC
CGY----ATTGTAACCTTCACCTATT
Annealed (N-terminal side)
LYFQSNA------
- --CTGTACTTCCAATCCAATGCX----------------
- - - GACATGAAGGTTAGGTTACGY........-.......
Fig. 7.2. LIC procedure using pMCSG vectors. All MCSG vectors contain an Sspl site (AATATT) positioned immediately after the sequence encoding the TEV protease recognition site. Cleavage with Sspl (a blunt cutter) followed by treatment with T4 DNA polymerase in the presence of only dGTP generates 15-base pair overhangs on both ends of the vector. PCR products must be generated using primers that begin with the complements of these overhangs followed by nucleotides required for LIC processing and proper expression. The sense primer must begin, TACTTCCAATCCAATGCC---,
where dashes indicate nucleotides identical to the target gene, and requires the nucleotides GCC to stop the action of
the polymerase and encode alanine in the correct reading frame. The antisense primer, TTATCCACTTCCAATGTTA---,
requires a G, complement of the C that stops the endonuclease activity of T4 polymerase, and TTA, the complement of a stop codon. Annealing to treated vector positions the TEV protease site adjacent to the gene in the correct reading frame. The resulting protein has the residues SNA appended to its N terminus after TEV cleavage. Primers conforming to these restrictions can be designed manually or using commercial programs or online tools such as the Express Primer Tool (14), http://tools.bio.anl.gov/bioJAVA/jsp/ExpressPrimerTool/.
restraints were placed on the design of the LIC region (3). The use of Sspl at the center of the LIC region allows the resulting proteins to carry only three additional amino acids on their N termini after proteolysis - serine from the TEV site, asparagine from the Sspl site, and alanine, the preferred choice for the limited number of amino acids possible due to the G used to stop the action of T4 polymerase in creating the overhang.
A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins
109
In addition to having the exact 16 base pairs at each end needed to create the complementary 15-base pair overhangs and stop the T4 polymerase, PCR primers must carry additional nucleotides (Fig. 7.2). For the sense primer, an additional two nucleotides are needed to maintain the proper reading frame and complete the codon begun with the required G. Because alanine is the most benevolent of the amino acids whose codons begin with G, the bases CC (or CA, CT, or CG) are added to give TACTTCCAATCCAATGCC. For the anti-sense primer, the required 16 nucleotides should be followed by the complement of a stop codon to prevent readthrough into vector sequences, giving TTATCCACTTCCAATGTTA. Following these sequences, primers terminate with nucleotides complementary to the gene being amplified, according to the requirements of the PCR conditions to be used. Primers may be designed manually or with software, such as the Express Primer tool (http://tools.bio.anl.gov/bioJAVA/jsp/ExpressPrimer-Tool/) (14).
Use of highly purified vectors is not essential to successful LIC, but improves efficiencies. However, complete cutting of the vectors with Sspl is critical and problematic. Simple restriction digests with commercial, normal-strength Sspl resulted in high backgrounds - clones containing unmodified vector free of an inserted gene (15). To circumvent this problem, vectors are cleaved with excess Sspl using high concentration, LIC-qualified commercial enzyme, then purified on agarose gels to remove traces of uncut vector. When processed in this fashion, we routinely achieve efficiencies between 75 and 90%.
7.2. Materials
7.2.1.1. PCR
7.2.1. PCR 1. Sense primer: 5'-TACTTCCAATCCAATGCC---and
Amplification of Genes Antisense primer: 5'-TTATCCACTTCCAATGTTA---
and Processing for LIC obtained from oligonucleotide production service of choice.
The dashes denote a series of nucleotides identical to those of the target gene (see Section 7.1 and (14))
2. Platinum Pfk DNA polymerase (Invitrogen, kit cat. no. 11708-013)
3. Platinum PJx lOx buffer (included in kit)
4. MgS04 (50mM) (included in kit)
7.2.1.2. Purification
1. Qiagen spin column (QIAquick PCR Purification Kit #28104)
2. Qiagen buffers (included in kit)
110
Eschenfeldt et al.
7.2.1.3. T4 Treatment
7.2.2. Preparation of Vector for LIC
7.2.2.1. Vector Prep
7.2.2.2. Sspl Treatment
7.2.2.3. T4 Treatment
7.2.3. LIC Annealing and Transformation
1. dCTP (lOOmM) (Promega cat. no. U1221)
2. Dithiothrcitol (DTT, 100 mM), molecular biology grade (Sigma cat. no. D-9779)
3. T4 DNA polymerase, LlC-qualified (Novagen cat. no. 70099)
4. lOx T4 polymerase buffer (included with polymerase)
1. LB Broth, Miller or other rich medium
2. LB Agar, Miller
3. Sterile polystyrene Falcon tube (14ml)
4. QIAGEN Plasmid Midi Kit (Qiagen, Inc. cat. no. 12143 or 12145)
5. Ampicillin
6. Chloramphenicol
7. Spectinomycin
8. Oakridge centrifuge tubes (Nalgene 3118-0050)
1. Sspl, Genome-Qualified, High Concentration, ca. 50U/ul (Promega R4604) and Sspl buffer (included with enzyme)
2. 50x TAE Buffer concentrate for gel: 2M Tris/Acetate, pH 8.0, 50mMEDTA
3. Ultrapure agarose for gel
4. Ethidium bromide, 50ug/ml
5. Tracking dye: 0.25% bromophenol blue plus 0.25% xylene cyanol FF in 30% glycerol
6. Gel electrophoresis apparatus - Biorad Mini Sub Cell or other comparable equip
7. DNA molecular weight markers, 1-kb ladder
8. QIAEX II gel extraction kit (Qiagen)
1. dGTP(lOOmM)
2. Dithiothrcitol (DTT, 100mM), molecular biology grade
3. T4 DNA polymerase, LIC-qualified (Novagen cat. no. 70099)
4. lOx T4 polymerase buffer (included with enzyme)
1. Sterile polystyrene Falcon tube (14 ml)
2. DH5ot cells (Invitrogen Library Competent, cat. no. 18263-012), or equivalent
3. SOC (included with DH5oc cells)
A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins
111
7.3. Methods
7.3.1. PCR and Preparation of PCR Product for LIC
7.3.1.1. PCR
7.3.1.2. Purification of PCR Product
1. In a thin-walled PCR tube combine 5ui lOx reaction buffer and primers at 1 |J.M with approximately 1 ng template DNA in a total volume of 48.5 ul.
2. Add 0.5 ul polymerase (1.25 U) and 0.5-1.5 \xl 50 mM MgS04 (as needed).
3. Perform PCR at appropriate temperatures for amplifying specific targets. Typical reactions comprise an initial 3-min dena-turation at 94°C, amplification through 35 cycles of 30s at 94°C, 45 s at 55-60°C, and 1 min at 68°C, with a final extension fori0min at 68°C (see Note 1).
1. Apply PCR to Qiagen Spin column from QIAquick PCR Purification Kit.
2. Follow the protocol detailed in the kit.
3. Determine concentration spectrophotometrically (e.g., Nan-oDrop Technologies' ND-1000 spectrophotometer) or by another suitable method.
7.3.1.3. T4 Polymerase Treatment of PCR Product
1. To a 0.4-ml Eppendorf tube on ice add
Volume
PCR product 20 ng	x\ü (x + y = 32 ul)	WKBm
T4 polymerase lOxreaction buffer	4ul	
dCTP(lOOmM)	lul	mm
DTT(lOOmM)	2ul	
Sterile water	yixl (* + ?-32ul)	tggmgmggffi : ■ ■
T4 DNA polymerase	lul	
KHHHHMMHHHNHHHflBHHflHHHH	Total 40 ul	
2. Incubate the reaction mix at room T for 30min
3. Inactivate the T4 DNA polymerase by heating at 75°C for 20 min
7.3.2. Preparation of Vector for LIC
7.3.2.1. Isolation and Purification of Vector
Inoculate 2 ml of LB broth containing the appropriate antibiotic in a 14-ml Falcon tube from a glycerol stock of E. coli DH5a containing the desired vector. Incubate for several hours at 37°C, agitating at 250rpm. Antibiotic concentrations: ampicillin, lOOug/ml; chloramphenicol, 30|0.g/ml; spectinomycin, 50ug/ml. (5^ Note 2).
112
Eschenfeldt et al.
2. Inoculate 50ml of LB plus antibiotic in a sterile, notched Erlenmeyer flask with the entire 2-ml culture and incubate overnight at 37°C at 250 rpm.
3. Transfer the culture to two 50-ml Oakridge centrifuge tubes and centrifuge at 6,000 x ß for 15 min at 4°C. Pour off supernatant fluid and invert the tubes to drain.
4. Lyse cells and purify vector using QIAGEN Plasmid Midi Kit according to the instructions detailed in the QIAGEN Plasmid Purification Handbook that accompanies the product.
5. Dissolve the pellet resulting from the purification in 200-500 ul water or 10 mM Tris (pH 8.0). The DNA concentration can be estimated by running an aliquot on an agarose gel or spectrophotometrically with a Nanodrop instrument (see Note 3).
7.3.2.2. Sspl Digestion 1. To a 1.5-ml Eppendorf tube on ice add
Volume
Vector DNA 15ug instrument	x ul (x + y -	52 ul)
Sspl lOx reaction buffer	6)il	
Sterile water	y ul (x + y =	52 ul)
Sspl	2ul	
Total 60ul		
2. Incubate for 2h at 37°C
3. Prepare a 50-ml, 0.8% TAE agarose gel containing ethidium bromide at 0.5ug/ml in a 6.5 x 10-cm flat bed electrophoresis tray fitted with an 8-well comb
4. Add lOul of 6x tracking dye to the digestion mixture
5. Load 10-15 ul per well
6. Run the gel at 75 V for approximately 2.5 h or until the molecular weight ladder is well separated
7. Visualize plasmid with a UV light box and excise band containing cut vector and transfer to tared 1.5-ml sterile Eppen-dorf tubes (see Note 4)
8. Extract DNA from gel slices using a QIAEX II Gel Extraction kit following the instructions detailed in the product's manual (see Note 5)
A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins 113
7.3.2.3. T4 Polymerase Treatment	1. To a 0.4-ml Eppendorf tube on ice add Volume	
	Vector DNA 200ng                                  X ul (x + y - 32 ul)	
	T4 polymerase lOx reaction buffer                 4 u.1	
	dGTP(lOOmM)                                      1 ul	
	DTT(lOOmM) 2ul	
	Sterile water                                             y ul (x + y = 32 ul)	
	Novagen T4 DNA polymerase                       1 ul	
	Total 40 ul	
	2. Incubate the reaction mix at room T for 30'	
	3. Inactivate the T4 DNA polymerase by heating at 75°C for 20' (see Note 6)	
7.3.2.4. Large-Scale Preparation of Vector for Microliter Plate Experiments	For stocks for several 96-well plates, all steps are scaled up 5 10-fold (see Note 7).	to
7.3.3. LIC Annealing and Transformation	1. In a 14-mI Falcon tube on ice mix	
of Cells	15 ng. vector DNA                                (ca. 3ul) ^flHHHH	
	30-45 ng PGR product DNA                (ca. 2-3 ul)	
7.3.3.1. LIC Annealing	Place the two aliquots together in a small droplet in the bottom of the tube in a total volume less than 8ui.	
	2. Incubate for 30min on ice	
7.3.3.2. Transformation	1. To the tube, add 50 ul of Invitrogen Library Competent DH5oc cells.	
2. Incubate on ice for 30 min.		
	3. Heat shock 45 s at 42°C.	
	4. Chill on ice 2 min.	
	5. Add 0.45 ml SOC.	
	6. Incubate at 37°C, 250rpm, lh.	
	7. Plate on LB agar containing the appropriate antibiotic(s). Antibiotic concentrations: ampicillin, 100ug/ml; chloram-	
phenicol, 30ug/ml; spectinomycin, 50ug/ml. Plate 100 ul of
114
Eschenfeldt et al.
the culture, then centrifuge the remaining 400 (al gently in an Eppcndorf tube to concentrate the cells. Decant the supernatant, resuspend the pellet in the small volume of medium remaining (ca. 50pl), and plate on a second LB agar plate.
7.4. Notes
1. Other proofreading DNA polymerases and PGR conditions may be used for amplification of genes. For high-throughput cloning into the MCSG vectors, PGR is routinely performed in 96-well plates using 1 U of KOD polymerase, lOng genomic target DNA, and primers at 0.2 uM in a 50-pl reaction.
2. If cultures are started in the morning, they should be slightly turbid by afternoon.
3. The DNA pellet usually is quite clear and difficult to see. Incubating the water/buffer over the position where the pellet is expected for several minutes to allow time for it to dissolve can improve yields moderately. Absorbance at 260 nm tends to overestimate the DNA concentration.
4. Compared to normal gel purifications, the gel is grossly overloaded and the DNA band often appears deformed. Run the gel out until the tracking dye approaches the end of the gel, so that the 5- to 6-kb vector band is well separated.
5. The agarose gel fragments should be cut into small pieces before extraction and can be stored overnight at 4°C prior to extraction if necessary. The extracted DNA can be frozen at -20°C if necessary.
6. Can store frozen, but precipitation will occur. Be sure to bring the solution to room temperature after storage and wait for cloudiness to clear.
7. To scale up preparation for cloning in microtiter plates (Chapter 8), start two 5-ml cultures of LB in the morning (inoculate heavily from glycerol stocks) and subculture into 500-ml medium for overnight incubation. Harvest cells in 250-ml centrifuge bottles and purify the vector using the QIAGEN Plasmid Maxi Kit, resuspending the final pellet in 0.5-ml water or buffer. This preparation typically yields about 500 ug of vector. For Sspl digestion, incubate 100-200 ug vector with 200-U Sspl for2h at 37°C in a volume of600 pi. Add 100-ul tracking dye and purify the cut vector on a 150-ml agarose gel in a 11 x 14-cm tray fitted with a 14-tooth comb, loading 50 pi per well. Combine gel fragments and elute the extract the DNA using the QIAEX II Gel Extraction kit according to the instructions provided by the vendor.
A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins
115
Acknowledgments
This work was supported by grants from the NIH (GM62414, GM074942), A. Joachimiak, PI, and the U.S. Department of Energy, Office of Biological and Environmental Research under Contract W-31-109-ENG-38.
References
1. Aslanidis, C. & de Jong, P. J. (1990) Ligation-independent cloning of PCR products (LIC-PCR), Nucleic Acids Res. 18, 6069-74.
2. Parks, T. D., Leuther, K. K., Howard, E. D., Johnston, S. A. & Dougherty, W. G. (1994) Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase, Anal. Biochem. 216, 413-7.
3. Stols, L., Gu, M., Dieckman, L., Raffen, R., Collart, F. R. & Donnelly, M. I. (2002) A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site, Protein Expr. Purif. 25,8-15.
4. Kapust, R. B. & Waugh, D. S. (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused, Protein Sei. 8, 1668-74.
5. Donnelly, M. I., Zhou, M., Millard, C. S., Clancy, S., Stols, L., Eschenfeldt, W. H., Collart, F. R. & Joachimiak, A. (2006) An expression vector tailored for large-scale, high-throughput purification of recombinant proteins, Protein Expr. Purif. 47, 446-54.
6. Nygren, P. A., Stahl, S. & Uhlen, M. (1994) Engineering proteins to facilitate bioprocess-ing, Trends Biotechnol. 12, 184-8.
7. Stols, L., Zhou, M., Eschenfeldt, W. H., Millard, C. S., Abdullah, J., Collart, F. R., Kim, Y. & Donnelly, M. I. (2007) New vectors for coexpression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols, Protein Expr. Purif. 53, 396-403.
8. Xu, Z., Horwich, A. L. & Sigler, P. B. (1997) The crystal structure of the asym-
metric GroEL-GroES-(ADP)7 chaperonin complex, Nature 388, 741-50. 9. Dieckman, L. J., Zhang, W., Rodi, D. J., Donnelly, M. I. & Collart, F. R. (2006) Bacterial expression strategies for human angio-genesis proteins, /. Struct. Funct. Genomics 7, 23-30.
10. Donnelly, M. I., Stevens, P. W., Stols, L., Su, S. X., Tollaksen, S., Giometti, C. & Joachimiak, A. (2001) Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone, Protein Expr. Purif. 22, 422-9.
11. Raines, R. T., McCormick, M., Van Oos-bree, T. R. & Mierendorf, R. C. (2000) The S.Tag fusion system for protein purification, Methods Enzymol. 326, 362-76.
12. Nallamsetty, S., Kapust, R. B., Tozser, J., Cherry, S., Tropea, J. E., Copeland, T. D. & Waugh, D. S. (2004) Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro, Protein Expr. Purif. 38, 108-15.
13. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990) Use ofT7 RNA polymerase to direct expression of cloned genes, Methods Enzymol. 185, 60-89.
14. Yoon, H. Y., Hwang, D. C, Choi, K. Y. & Song, B. D. (2000) Proteolytic processing of oligopeptides containing the target sequences by the recombinant tobacco vein mottling virus NIa proteinase, Mol. Cells 10, 213-9.
15. Dieckman, L., Gu, M., Stols, L., Donnelly, M. I. & Collart, F. R. (2002) High throughput methods for gene cloning and expression, Protein Expr. Purif. 25, 1-7.