Bio-Rad Profinity eXact Purification Resin User Manual

For Technical Support, call your local Bio-Rad office, or in the U.S.,

call 1-800-4BIORAD (1-800-424-6723).

Profinity eXact

™

Protein

Purification System

Instruction Manual

Table of Contents

Section 1 Introduction............................................................1

1.1 Introduction ......................................................................1

Section 2 Product Description ..............................................3

2.1 Profinity eXact System Components ................................3

2.2 Profinity eXact Expression Technology ............................4

2.3 Profinity eXact Purification Technology ............................4

2.4 Resin Characteristics ......................................................6

2.5 Chemical Compatibility ....................................................6

Section 3 Profinity eXact System Strategies ......................10

Section 4 Cloning and Expression Procedures..................12

4.1 pPAL7 Expression Vector ..............................................12

4.2

E. coli

C-Max5a Cloning Host Cell ................................12

4.3

E. coli

BL21(DE3) Expression Host Cell ........................12

4.4 Cloning Region Sequence ............................................14

4.5 General Cloning Guidelines ..........................................14

4.6 Cloning with the Linearized, RIC-Ready pPAL7 Vector ..15

4.6.1

RIC Cloning

........................................................15

4.6.2

Restriction Enzyme Digestion Cloning

................18

4.7 Cloning with the Supercoiled pPAL7 Vector ..................20

Section 5 Protein Expression and Lysate Preparation ......22

5.1 Protein Expression ........................................................22

5.2 Quick Solubility Screening Protocol................................23

5.3 Lysate Preparation ........................................................25

5.3.1

Lysis/Cell Resuspension Buffer Selection (NaCI, Tris-HCI, and pH)

....................................25

5.3.2

Nucleases and MgCI

........................................26

5.3.3

Protease Inhibitors

..............................................26

5.3.4

Urea and Guanidine-HCI

....................................26

5.3.5

Profinity eXact MBP Control Lysate

....................27

5.3.6

Lysis Protocol

......................................................27

Section 6 Purification Procedures ......................................28

6.1 General Guidelines ........................................................28

6.1.1

Wash Buffer Selection

........................................28

6.1.2

Lysate Loading

....................................................29

6.1.3

Profinity eXact MBP Control Lysate

....................29

6.1.4

Cleavage and Elution

..........................................30

6.1.5

Cleaning and Regeneration

................................30

6.2 Profinity eXact Mini Spin Column Kit Purifications ..........30

6.3 Bio-Scale Mini Cartridge Purifications With

LC Instrumentation ........................................................32

6.4 Bio-Scale Mini Cartridge Purifications With Syringe........34

6.5 Gravity Column Purifications ..........................................36

Appendix A N-terminal Target Protein Structure and

Cleavage Kinetics ..............................................38

Appendix B Intrinsic Cleavage Rates....................................41

Appendix C Triggered Cleavage Rates ................................42

Appendix D Comprehensive Chemical Compatibility List ..44

Appendix E Troubleshooting Guide......................................47

Appendix F References ........................................................49

Appendix G Ordering Information ........................................51

Appendix H Legal Information ..............................................52

Section 1 Introduction

Fig. 1. One-step tag cleavage and elution: Pure and simple.

1.1 Introduction

Affinity tags are commonly used to facilitate the purification of recombinant proteins. Once purified, these proteins are often studied to further elucidate the protein’s structure, function, and interaction with other molecules. However, just as a protein’s intermolecular relationship with other proteins is complex, so is the intramolecular dynamic between a fusion protein’s affinity tag and the target protein. An affinity tag or protease recognition sequence may negatively interfere with the protein’s natural biochemical properties or characteristics (1, 2, 4, 5, 7–12, 15, 16, 18), yielding ambiguous experimental results. Consequently, researchers have utilized a variety of methods to remove the affinity tag from a purified protein.

The current technology for tag removal is tedious and time consuming, requiring a number of steps. After initial purification, the eluted tagged protein is exposed to a cleavage enzyme, which hydrolyzes the tag from the protein fusion. A subsequent step is then needed to isolate the protein from the tag and the protease, which can result in significant loss of product, additional investment of labor, and increased cost to the overall purification.

The Profinity eXact™(exact affinity cleavage technology) protein purification system offers a novel alternative to existing affinity purification and cleavage tools (Figure 1). The system utilizes an immobilized, extensively engineered protease that both recognizes and avidly binds (KD< 100 pM) to the small N-terminal co-expressed affinity tag in the protein fusion. Subsequent to column washing, the protease performs a specific, controlled cleavage and removal of the tag from the fusion directly on the column, resulting in the

release of highly purified recombinant protein with a native N-terminus. During elution, the cleaved Profinity eXact tag remains tightly associated with the resin’s immobilized protease, eliminating the need for additional steps to remove the protease and the tag. Addressing a key dissatisfaction with recombinant tag technology, the Profinity eXact system improves the efficiency of tag removal by generating a highly purified protein containing only its native amino acid sequence—all with one simple elution step (see Figure 2). The tag-free protein’s crystal structure, protein function, and interactions with other proteins may be studied without concern for an interfering affinity tag. Additionally, this purification system is well suited for the expression of peptides, whose small size often complicates expression and purification.

Fig. 2. Profinity eXact affinity-tagged maltose binding protein (MBP) purified with a 1 ml Bio-Scale Mini™Profinity eXact cartridge on a Biologic Duoflow™system. Samples

were run on a 4–12% Criterion™Bis-Tris gel. Lanes 1 and 6, Precision Plus Protein™standards; lane 2, crude

E. coli

extract; lane 3, host protein contaminants in the flow-through fraction; lane 4, host protein contaminants washed from the cartridge by Profinity eXact bind/wash buffer; and lane 5, eluted tag-free MBP.

Section 2 Product Description

2.1 Profinity eXact™ System Components

Table 1. Profinity eXact System Components and Storage Conditions.

CClloonniinngg aanndd EExxpprreessssiioonn SSttaarrtteerr KKiitt,, 2200 rreeaaccttiioonnss CCaattaalloogg NNoo.. 115566--33000000 pPAL7 RIC-ready vector 0.5 µg –20°C

pPAL7 supercoiled vector 10 µg –20°C SOC medium 2 × 10 ml Ambient/–70°C

E. coli

BL21(DE3) chemical competent expression cells 10 × 50 µl –70°C

E. coli

C-Max5a chemical competent cloning cells 10 × 50 µl –70°C

pUC19 control (10 ng/µl) 10 µl –70°C RRIICC--RReeaaddyy EExxpprreessssiioonn VVeeccttoorr,, 2200 rreeaaccttiioonnss CCaattaalloogg NNoo.. 115566--33000011 pPAL7 RIC-ready vector 0.5 µg –20°C SSuuppeerrccooiilleedd EExxpprreessssiioonn VVeeccttoorr,, 2200 rreeaaccttiioonnss CCaattaalloogg NNoo.. 115566--33000022 pPAL7 Supercoiled vector 10 µg –20°C

EE.. ccoollii

BBLL2211((DDEE33)) EExxpprreessssiioonn CCoommppeetteenntt CCeellllss KKiitt,, 1100 rreeaaccttiioonnss CCaattaalloogg NNoo.. 115566--33000033 BL21(DE3) expression cells 10 × 50 µl –70°C pUC19 control (10 ng/µl) 10 µl –20°C /–70°C SOC medium 1 × 10 ml Ambient/–70°C

MMoonnoocclloonnaall AAnnttiibbooddyy CCaattaalloogg NNoo.. 115566--33000044 Profinity eXact affinity-tagged antibody 1 vial –20°C BBoottttlleedd RReessiinn CCaattaalloogg NNoo.. 115566--33000055 Profinity eXact resin 10 ml 4°C MMiinnii PPuurriiffiiccaattiioonn SSttaarrtteerr KKiitt,, 1100 ppaacckk CCaattaalloogg NNoo.. 115566--33000066

Profinity eXact mini spin columns 10 each 4°C 2 mL capped collection tubes 50 each Lyophilized Profinity eXact MBP control lysate 1 vial 4°C Bacterial lysis and extraction reagent 10 ml ambient Binding/washing buffer 50 ml 4°C/ambient Elution buffer 20 ml 4°C/ambient

MMiinnii SSppiinn CCoolluummnnss,, 1100 ppaacckk CCaattaalloogg NNoo.. 115566--33000077 Profinity eXact mini spin columns 10 pack 4°C 2ml capped collection tubes 50 pack

PPuurriiffiiccaattiioonn CCaarrttrriiddggeess,, 22 ppaacckk oorr 44 ppaacckk CCaattaalloogg NNooss.. 773322--44664466

&& 773322--44664477 Profinity eXact cartridges, 1 ml 2 or 4 4°C PPuurriiffiiccaattiioonn CCaarrttrriiddggee,, 11 ×× 55 mmll CCaattaalloogg NNoo.. 773322--44664488 Profinity eXact cartridges, 5 ml 1 4°C EExxpprreessssiioonn aanndd PPuurriiffiiccaattiioonn SSttaarrtteerr KKiitt CCaattaalloogg NNooss.. 115566--33000000

&& 115566--33000066 Cloning and expression starter kit 20 reactions –70°C Mini purification starter kit 10 each 4°C

2.2 Profinity eXact Expression Technology

Fusion proteins with an N-terminal Profinity eXact tag are expressed with the pPAL7 expression vector (5.9 kb). This inducible expression vector utilizes the strong, tightly regulated T7 lac promoter (6). The pPAL7 plasmid confers ampicillin resistance, constitutively expresses the lacI repressor, and has been designed to facilitate cloning of a target gene through several different methods.

Precise fusions—in which the resultant purified product does not contain any additional amino acids at the N-terminus—may be constructed using the HindIII restriction site and an appropriate restriction site in the multiple cloning site (MCS) or by cloning with the prepared restriction-independent cloning (RIC) vector. Use of the linearized, RIC-ready vector facilitates high-throughput cloning of DNA sequences, regardless of the restriction sites that may be present within a target gene.

Some proteins exhibit significant N-terminal structure that may affect binding to the resin (Appendix A: N-terminal Target Protein Structure and Cleavage Kinetics), or their first two amino acids result in non-ideal cleavage kinetics (Appendix B: Intrinsic Cleavage Rates and Appendix C: Triggered Cleavage Rates). The challenges presented by these proteins are easily addressed at the cloning stage by utilizing a two amino acid linker (such as the recommended Thr-Ser linker) between the Profinity eXact tag and the target protein—generating an

imprecise fusion

. A Thr-Ser spacer between the Profinity eXact tag and the target protein ensures optimal cleavage kinetics and purification, regardless of the target protein. These imprecise fusion proteins, once purified, retain the Thr-Ser linker at the N-terminus.

2.3 Profinity eXact Purification Technology

The Profinity eXact purification resin utilizes Superflow™crosslinked agarose coupled to a unique subtilisin mutant. The Superflow support matrix exhibits low nonspecific protein binding while providing the high mechanical stability and flow dynamics desired for medium pressure liquid chromatography applications (see Table 2).

Mutant subtilisin protease—S189, the functional ligand immobilized to the support matrix—has been precisely engineered for affinity chromatography (3, 14). S189 subtilisin is extremely stable and efficiently refolds

in vitro

The recombinant protease also is designed to specifically recognize and

cleave at the C-terminus of a nine amino acid sequence (EEDKLFKAL). Through a third set of mutations, this cleavage reaction has been changed from a self-initiating digestion to one that is triggered by 100 mM potassium fluoride; complete cleavage is most often achieved within 30 min, at room temperature.

The subtilisin exhibits extremely high binding affinity to the 8 kD Profinity eXact tag (KD< 100 pM). The Profinity eXact tag is a form of the wild-type subtilisin BPN' prodomain—genetically engineered for high stability and to contain the distinct cleavage recognition sequence of the subtilisin mutant. Additionally, the subtilisin prodomain serves as an exo-recognition sequence for the immobilized subtilisin, which significantly increases the system’s binding affinity and cleavage specificity to the tag.

Once it has been captured by the Profinity eXact purification resin—and the cleavage reaction has been triggered with a sodium fluoride-containing buffer—the affinity tag is precisely cleaved at the C-terminus of the cleavage recognition sequence. The result is a quickly purified protein product that lacks any amino acid residues derived from the Profinity eXact tag.

2.4 Resin Characteristics

The resin attributes are summarized in Table 2.

Table 2. Characteristics of Profinity eXact Purification Resin.

Functional ligand Mutant subtilisin, S189

Base bead Superflow agarose (6% crosslinked)

Format Spin column: 600 µl 17% suspension

(100 µl settled resin)

Bottled: 50% suspension

Cartridge: 1 or 5 ml packed resin

Storage buffer: 100 mM sodium phosphate,

0.02% sodium azide, pH 7.2

Particle size 60–160 µm

Molecular exclusion limit 6,000 kD

Recommended linear flow rate <1,000 cm/hr at 25°C*

pH stability 2–13

Chemical compatibility See Table 4 and Appendix D: Comprehensive

Chemical Compatibility List

Storage 4°C

Shelf life in storage buffer When unopened, 1 year from receipt; store at 4°C

Operational temperature 4–40°C

* Recommended linear flow rate determined by the following factors:

The flow rate of 100 mM sodium phosphate, pH 7.2 was incrementally increased through a Bio-Scale Mini Profinity eXact 1 ml cartridge packed with a 20% compression factor. The pressure-flow curve for the Profinity eXact purification resin became nonlinear at linear velocities above 1,000 cm/hr.

2.5 Chemical Compatibility

The Profinity eXact purification system is compatible with a broad range of additives commonly used to purify proteins. Extensive information is listed in Appendix D: Comprehensive Chemical Compatibility List.

Highest purification yields are achieved by eliminating Cl–ions from all lysis and wash buffers. The chloride ions from additives like NaCl,

KCl, and Tris-HCl act as slower cleavage/elution-triggering anions (see Table 3). NaCl or KCl salt is commonly added to lysis and wash buffers to reduce electrostatic interactions between background proteins (or DNA) with a chromatographic support. When using the Profinity eXact purification resin, sodium acetate (NaOAc) and potassium acetate (KOAc) are

recommended substitutes for NaCl or KCl. If using the 0.1 M sodium phosphate Profinity eXact bind/wash buffer, a higher sodium phosphate concentration (e.g. 0.3–1.0 M, pH 7.2) may also be used to raise the ionic concentration of the lysis and wash buffers.

Table 3. Triggering Anions.

Triggering Compound Concentration for Concentration for

Anion Fast Cleavage Moderate Cleavage

–

NaF, KF 100 mM 5 mM

–

NaN

10 mM 1 mM

–

NaNO

5 mM 1 mM

HCO

–

NaHCO

1,000 mM 25 mM

–

NaCl, KCl >1,000 mM 75 mM

Although the wild-type subtilisin BPN' is a serine protease, the purification resin is unaffected by most serine protease inhibitors due to the Profinity eXact subtilisin’s active site mutations. The protease cocktails tested include combinations of the following inhibitors: AEBSF, aprotinin, benzamidine, E-64, EDTA, leupeptin, pepstatin A, phenanthroline, and PMSF. See Table 4 for additional information regarding the compatibility of protease inhibitors, common additives, and solutions typically used in affinity chromatography methods.

Table 4. Chemical Compatibility (Partial List—See Appendix A for Complete List)

RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraattiioonn

Triggering Ions F–, Cl–, N

–

, NO

–

, HCO

–

Triggers cleavage of fusion Remove from all

llyyssiiss aanndd wwaasshh and elution of target buffers for highest yields protein See

TTaabbllee

3 for ionic concentrations

to use as trigger in elution buffer

Salts Sodium acetate Reduces non-specific protein £3 M (an excellent substitute for NaCl

binding due to ionic interactions in lysis and wash buffers)

NNaaCCll oorr KKCCll

Triggers cleavage of fusion Remove from all

llyyssiiss aanndd wwaasshh and elution of target protein buffers—use sodium acetate or

sodium phosphate instead to reduce electrostatic interactions

Buffers Tris-HCl Cl

–

in lysis and wash buffers Substitute with Tris-acetate or triggers cleavage of fusion Tris-phosphate and adjust pH with and elution of target protein acetic or phosphoric acid

Tris-acetate 50 mM tested Tris-phosphate 50 mM tested

Acids HCl Cl

–

in lysis and wash buffers Avoid use of HCl for highest yields; triggers cleavage of fusion instead, use acids such as and elution of target protein phosphoric or acetic acid

Detergents Non-ionic (NP-40, Triton

Solubilize proteins £5% (v/v)

X-100, Tween

-20)

Zwitterionic (CHAPS, Solubilize proteins £5% (v/v or w/v) CHAPSO, Dodecyl-b, D-maltoside, Octylthioglucoside)

Ionic (CTAB, Sarkosyl, Significantly lower yields Remove from all buffers SDS)

Protease PMSF F

–

triggers cleavage of fusion 0.5 mM; no adverse effects when

Inhibitors and elution of target protein incubated with resin for up to 3 hr

TLCK 100 µM TPCK 100 µM

Pharmingen™ Protease F

–

may lower yield slightly 2×

Inhibitor Cocktail

Calbiochem

Protease 1×

Inhibitor Cocktail Set I

RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraattiioonn

Roche cOmplete Protease F–may lower yield slightly 1× Inhibitor Cocktail Tablet

Lysis Solutions Bacterial Lysis and 1×

Extraction (Bio-Rad)

B-PER

in phosphate 1×

buffer (Pierce)

B-PER

(Pierce) Cl–from Tris-HCl results in 1×

slightly lower yield

ReadyPreps

™

Lysis Do not use

solutions (Epicentre)

BugBuster

(EMD) 1×

FastBreak

™

Cell Lysis 1×

(Promega)

CelLytic

™

Express Do not use

(Sigma)

Denaturants Guanidine•HCl Cl

triggers cleavage of fusion Remove from all

llyyssiiss

and

wwaasshh

during load and wash steps buffers

Urea Solubilizes proteins £2 M; with 4 M, ~30% reduced

control MBP binding capacity observed; with 8 M, ~70% reduced control MBP binding capacity and reduced purity observed

Other Additives CaCl

Essential component for £5 mM; use with buffer such as purification of Ca

-binding MES, MOPS, or PIPES buffer to

proteins eliminate precipitation of Ca

with

either fluoride- or phosphate-

–

triggers cleavage during containing Profinity eXact buffers load and wash steps

MgCl

Essential metal cofactor for £5 mM; if higher Mg

nucleases concentrations are desired, use

magnesium acetate instead

–

triggers cleavage during load and wash steps Do not add to Profinity eXact elution

buffer—Mg

precipitates with F–; instead, use an elution buffer with an alternative trigger, such as 10 mM NaN

, with 100 mM sodium

phosphate, pH 7.2

Section 3 Profinity eXact

™

System Strategies

1. Basic purification strategy—create and purify an imprecise fusion that ensures initial purification success with minimal optimization by incorporating a Thr-Ser spacer.

a. Determine cloning method and construct fusion protein

RIC-ready pPAL7 (Section 4.6)

Supercoiled pPAL7 (Section 4.7)

b. Express Profinity eXact™affinity-tagged fusion protein

E. coli

culturing and expression (Section 5.1)

Solubility check of expressed fusion protein (Section 5.2)

c. Lyse cells and store lysate at 4°C

Bacterial lysis and extraction reagent (provided with mini spin volumn purification kit)

Cell resuspension buffer (100 mM sodium phosphate, pH 7.2) and lyse using desired lysis method

d. Purify protein with chloride-free wash (100 mM sodium phosphate,

pH 7.2; 4°C) and elution (100 mM sodium fluoride, 100 mM sodium phosphate, pH 7.2; room temperature) buffers using appropriate purification protocol

Spin column (Section 6.2)

Bio-Scale™Mini cartridge with LC instrument (Section 6.3)

Bio-Scale Mini cartridge with syringe (Section 6.4)

Gravity column (Section 6.5)

2. Precise purification strategy—create a precise fusion to obtain a completely tag-free, purified protein

a. Consider, if known, the P1' and P2' (first two N-terminal amino

acids of target protein) residues’ effect on cleavage kinetics

Appendix A: N-terminal Target Protein Structure and Cleavage Kinetics

Appendix B: Intrinsic Cleavage Rates

Appendix C: Triggered Cleavage Rates

b. Determine cloning method and construct fusion protein

RIC-ready pPAL7 (Section 4.6)

Supercoiled pPAL7 (Section 4.7)

c. Express Profinity eXact™affinity-tagged fusion protein

E. coli

culturing and expression (Section 5.1)

Solubility check of expressed fusion protein (Section 5.2)

d. Lyse cells and store lysate at 4°C

Select appropriate chloride-free lysis buffer (Section 5.3.1)

Lyse using desired lysis method (Section 5.3.6)

e. Identify appropriate buffers and conditions

Select appropriate chloride-free wash buffer (Section 6.1.1)

f. Purify protein using appropriate purification protocol

Spin column (Section 6.2)

Bio-Scale Mini cartridge with LC instrument (Section 6.3)

Bio-Scale Mini cartridge with syringe (Section 6.4)

Gravity column (Section 6.5)

Section 4 Cloning and Expression Process

4.1 pPAL7 Expression Vector

The pPAL7 expression vector utilizes the T7lac promoter to strongly express a Profinity eXact fusion protein in

E. coli

cells producing T7 RNA polymerase. The pPAL7 plasmid constitutively expresses the lacI repressor and confers ampicillin resistance to the host cell (Figure 3). The Profinity eXact™tag is an 8.2 kD affinity tag that functions as an N-terminal fusion tag (Figure 3).

4.2

E. coli

C-Max5aaCloning Host Cell

The

E. coli

C-Max5a cells [F–j80 dlacZDM15 D(

lac

ZYA-

arg

F)U169

rec

endA1

hsd

R17(r

–

, m

)

phoA sup

E44 l

–

thi

-1

gyr

A96 relA1] are chemical competent transformation cells that are specifically designed to maximize cloning success. This

E. coli

K12 derivative facilitates isolation of high-quality plasmid DNA and is an ideal host strain for transformation of ligation cloning reactions.

4.3

E. coli

BL21(DE3) Expression Host Cell

E. coli

BL21(DE3) cells [

E. coli

B F

dcm ompT hsd

S(r

–

, m

–

)

gal l(DE3)

] are used for high-level protein expression with T7 RNA polymerase-based expression systems. The strain is a derivative of

E. coli

B, which is deficient

in the

lon

protease as well as the

ompT

outer membrane protease,

facilitating the isolation of intact recombinant proteins. The host is a lysogen of lDE3 and contains the T7 RNA polymerase gene—integrated into the chromosome—under control of the lacUV5 promoter. 1–2 mM isopropyl-b-D-thiogalactopyranoside (IPTG) is used to induce the expression of recombinant proteins cloned into vectors downstream of a T7lac promoter and transformed into the

E. coli

BL21(DE3) cells.

Fig. 3. pPAL7 vector map.

Table 5. Features of the pPAL7 Expression Vector.

Vector Feature Vector Position

lac

promoter 1–17 Profinity eXact tag 92–316 MCS

SpeI 318 NcoI 325 BamHI 333 EcoRI 345 XhoI 351 NotI 358

T7 terminator 413–460

bla

(AmpR) 1,079–1,936

Ori

2,499

lacI

4,634–5,713

4.4 Cloning Region Sequence

Fig. 4. Sequence of Profinity eXact tag and cloning region.

4.5 General Cloning Guidelines

The pPAL7 vector is provided in both linearized and supercoiled form in the Profinity eXact cloning and expression starter kit. The linearized plasmid (Profinity eXact RIC-ready pPAL7 vector) may be ligated with PCR products possessing compatible overhangs generated by either the restriction-independent cloning (RIC) or traditional restriction enzyme digestion method. Both the RIC-ready and the supercoiled pPAL7 vector may be used to generate either precise- or imprecise-fusion proteins:

Precise fusions

—purifications result in a tag-free target protein without extra amino acids after cleavage of the Profinity eXact tag.

Imprecise fusions

—purifications result in the retention of a spacer (e.g., Thr-Ser) at the N-terminus of the target protein. Presence of the Thr-Ser spacer ensures successful purifications with minimal optimization.

T7 Promoter lac Operator -

1 TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATTATGCTGA GTGATATCCC CTTAACACTC GCCTATTGTT AAGGGGAGAT CTTTATTAAA ACAAATTGAA ATTCTTCCTC

Profinity eXact Tag (92-316) -

NdeI

GlyGlyLys SerAsnGlyGlu LysLysTyr IleValGly PheLysGlnGly PheLysSer CysAlaLys

81 ATATACATAT GGGAGGGAAA TCAAACGGGG AAAAGAAATA TATTGTCGGG TTCAAACAGG GCTTTAAGAG CTGCGCTAAG TATATGTATA CCCTCCCTTT AGTTTGCCCC TTTTCTTTAT ATAACAGCCC AAGTTTGTCC CGAAATTCTC GACGCGATTC

Profinity eXact Tag (92-316) -

LysGluAspVal IleSerGlu LysGlyGly LysLeuGlnLys CysPheLys TyrValAsp AlaAlaSerAla ThrLeuAsn·

161 AAGGAGGATG TCATTTCTGA AAAAGGCGGG AAACTCCAAA AGTGCTTCAA ATATGTAGAC GCAGCTAGCG CTACATTAAA TTCCTCCTAC AGTAAAGACT TTTTCCGCCC TTTGAGGTTT TCACGAAGTT TATACATCTG CGTCGATCGC GATGTAATTT

Profinity eXact Tag (92-316) -

SapI

Cleavage Recognition Sequences -

·GluLysAla ValGluGluLeu LysLysAsp ProSerVal AlaTyrValGlu GluAspLys LeuPheLys AlaLeuThrSer·

241 CGAAAAAGCT GTAGAAGAAT TGAAAAAAGA TCCGAGCGTC GCGTACGTAG AAGAAGACAA GCTCTTCAAA GCTTTGACTA GCTTTTTCGA CATCTTCTTA ACTTTTTTCT AGGCTCGCAG CGCATGCATC TTCTTCTGTT CGAGAAGTTT CGAAACTGAT

Profinity eXact Cleavage Site

NcoI

SpeI

·SThrMetAla GlySerGly CysGluPheLeu GluAlaAla Ala***

321 GTACCATGGC GGGATCCGGC TGCGAATTCC TCGAGGCGGC CGCATAAGCC CGAAAGGAAG CTGAGTTGGC TGCTGCCACC CATGGTACCG CCCTAGGCCG ACGCTTAAGG AGCTCCGCCG GCGTATTCGG GCTTTCCTTC GACTCAACCG ACGACGGTGG

T7 Terminator

401 GCTGAGCAAT AACTAGCATA ACCCCTTGGG GCCTCTAAAC GGGTCTTGAG GGGTTTTTTG CTGAAAGGAG GAACTATATC CGACTCGTTA TTGATCGTAT TGGGGAACCC CGGAGATTTG CCCAGAACTC CCCAAAAAAC GACTTTCCTC CTTGATATAG

XhoI -

BamHI EcoRI NotI STOP

HindIII SpeI

Optimal room-temperature purification of the target protein with the Profinity eXact resin is dependent upon the amino acid residues in the first two positions of the target protein. (Residues 1 and 2 are referred to as positions P1' and P2', respectively.) Target proteins with a P1' proline are not subject to resin cleavage and elution; on the other hand, proteins with a P1' cysteine have a particularly high intrinsic cleavage and elution rate with the Profinity eXact resin, even in the absence of a fluoride-containing elution buffer. Refer to Appendices A-C for more details. Target proteins with these challenging P1'-P2' amino acid residues should be cloned with a threonine-serine spacer. This spacer maximizes successful purification results, regardless of the target protein’s P1'-P2' residues or any effect its N-terminal structure may have on the Profinity eXact tag’s resin-binding properties.

If the presence of an additional Thr-Ser dipeptide (208 Da) at the N-terminus of the target protein is not an issue, cloning of a target gene and the spacer into the vector is the recommended strategy—requiring the least amount of purification optimization (either using the SpeI cloning site or obtaining a PCR target gene product with the Thr-Ser spacer at the N-terminus).

4.6 Cloning With the Linearized, RIC-Ready pPAL7 Vector

4.6.1 RIC Cloning

Because this cloning method is completely free of any use of restriction enzymes, it is independent of restriction enzyme sites within the target protein and is thus ideal for high-throughput cloning. The RIC-ready vector has been prepared to generate the

dephosphorylated

overhangs

depicted in Figure 5.

Fig. 5. RIC-ready pPAL7 vector.

AAGCTCTTCA HO-AATTCCTCGAGG TTCGAGAAGTTTC-OH GGAGCTCC

ready pPAL7

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