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

2

........................................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

1

2

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

3

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

4

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.

5

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

6

!

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

F

–

NaF, KF 100 mM 5 mM

N

3

–

NaN

3

10 mM 1 mM

NO

2

–

NaNO

2

5 mM 1 mM

HCO

2

–

NaHCO

2

1,000 mM 25 mM

Cl

–

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.

7

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

RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraattiioonn

Triggering Ions F–, Cl–, N

3

–

, NO

2

–

, HCO

2

–

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

8

!

!

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

2

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

2+

-binding MES, MOPS, or PIPES buffer to

proteins eliminate precipitation of Ca

2+

with

either fluoride- or phosphate-

Cl

–

triggers cleavage during containing Profinity eXact buffers
load and wash steps

MgCl

2

Essential metal cofactor for £5 mM; if higher Mg

2+

nucleases concentrations are desired, use

magnesium acetate instead

Cl

–

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

buffer—Mg

2+

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

3

, with 100 mM sodium

phosphate, pH 7.2

9

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

10

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)

11

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

A1

endA1

hsd

R17(r

k

–

, m

k

+

)

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

B

–

, m

B

–

)

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.

12

Fig. 3. pPAL7 vector map.

Table 5. Features of the pPAL7 Expression Vector.

Vector Feature Vector Position

T7

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

13

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.

14

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.

15

AAGCTCTTCA HO-AATTCCTCGAGG
TTCGAGAAGTTTC-OH GGAGCTCC

ready pPAL7

-