the amount of thrombin and the
`In addition to varying the time of incubation,
`temperature of incubation (up to 37°C) may also be varied to determine the optimum
`conditions for cleavage of a particularfitsion protein.
`
`ENZYMATIC CLEAVAGE OF MATRIX-BOUND GST FUSION PROTEINS
`
`In this alternate protocol, GST fusion proteins that contain a thrombin cleavage site are
`bound to glutathione-agarose as described in UNIT 16.7. Prior to elution feom the matrix,
`thrombin is added and the protein of interest is cleaved from the GST carrier. The cleaved
`protein is collected in the wash buffer and the GST carrier remains bound to the beads,
`permitting easy and efficient physical separation of the reaction products.
`
`ALTERNATE
`PROTOCOL 2
`
`Additional Materials
`
`For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
`solutions, see APPENDIX 2; for suppliers, see APPENDIX 4.
`
`GST fusion protein bound to glutathione-agarose beads (UNIT 16.7)
`1% (v/v) Triton X-100 in phosphate—buffered saline (PBS; APPENDIX 2)
`GST wash buffer: 50 mM Tris«Cl (pH 7.5)/150 mM NaCl
`GST elution buffer: 50 mM Tris-Cl (pH 8.0)/5 mM reduced glutathione
`20- or 50- ml screw-cap tube
`
`1. Wash GST fusion protein bound to glutathione-agarose beads with 20 vol of 1%
`Tréton X-100 in PBS, using a 20- or 50-ml screw-cap tube. Centrifuge
`sec in a
`tabletop centrifuge at 500 x g, room temperature, to pellet the beads. Carefully remove
`and discard the supernatant. Resuspend the beads in 20 vol Triton X-100 buffer and
`repeat wash.
`
`2. After the second centrifugation, carefully remove and discard the supernatant.
`Resuspend the beads in 20 vol GST wash buffer.
`
`3. Pellet the beads and discard the supernatant. Resuspend the beads in 20 vol thrombin
`cleavage buffer. Repeat the centrifugation and resuspend the beads in S1 ml thrombin
`cleavage buffer.
`
`Although it is easier to wash the beads in large volumes, the amount of thrombin
`cleavage buffer to use in the cleavage reaction is best kept to a minimum.
`
`4. Remove a small aliquot of resuspended beads and add an equal volume of 2x SDS
`sample buffer. Store at —20°C until analyzed by SDS-PAGE (step 7).
`
`This sample is used to estimate the amount offitsion protein bound to the beads.
`
`5. Add thrombin to the remaining bead slurry at a ratio of 1% (W/w) thrombin to the
`estimated amount of bound fasion protein. Incubate 1 hr at 25°C.
`
`As in solution cleavage, the amount ofthrombin. time, and temperature of incubation
`can be adjusted to optimize the cleavage efficiency.
`
`6. Elute the cleaved and released protein by washing the beads with 1 bed volume of
`GST wash buffer. Centrifuge as in step 1 to pellet beads and collect supernatant.
`Repeat five times, but keep each wash fraction separate. Remove 20—|.Ll aliquots from
`each wash fraction for SDS-PAGE.
`
`7. Elute bound GST by repeating step 6 with GST elution buffer instead of GST wash
`buffer. Remove 20~ul aliquots from each fraction and analyze by SDS-PAGE to
`determine extent of cleavage. Include the aliquot of beads from step 4 on this gel.
`
`Current Protocols in Molecular Biology
`
`Protein
`Expression
`
`16.4.9
`
`Supplement 28
`
`BEQ 1016
`Page 190
`
`

`
`If cleavage is incomplete, the time of incubation and/or the amount of enzyme can be
`increased.
`
`Because this is an analytical-scale experiment, it is easiest to discard these beads after
`one use. Upon scaling up the procedure, the beads can be regenerated, as described
`in Reagents and Solutions, UNIT 16. 7.
`
`ALTERNATE
`PROTOCOL 3
`
`ENZYMATIC CLEAVAGE OF FUSION PROTEINS WITI-E
`ENTEROKINASE
`
`Enterokinase (also called enteropeptidase) is a mammalian trypsin-like serine protease
`that displays a high degree of specificity for the sequence (Asp)4-Lys, cleaving on the
`carboxy-terminal side of the lysine residue of the recognition sequence. Although in
`mammals the enzyme has evolved to recognize and cleave this sequence from the
`amino-termini of trypsinogens, it has been shown that enterokinase is also capable of
`cleaving fusion proteins that are expressed in bacteria and that contain this recognition
`sequence inserted between the carrier protein and the carboxy-terminal fusion partner.
`Enterokinase is capable of cleaving fusion proteins under a wide range of reaction
`conditions, with pH ranging from 4.5 to 9.5 and temperatures ranging from 4° to 45°C.
`Enterokinase is also extremely tolerant of the nature of the amino acid residue in the P1’
`position (except that the peptide bond between Lys-Pro at this position is totally refractory
`to cleavage; E. LaVallie and L. liacie, unpub. observ.). At sufficiently low ionic strength,
`enterokinase can cleave fusion proteins at a weight ratio of 1 2500 to 122000. At these ratios,
`typical cleavage reactions are carried out for 16 to 24 hr at 37°C, but these parameters
`(time, temperature, and enzyme/substrate ratio) can be adjusted as needed.
`
`The thioredoxin fusion vector pTRXFUS (UNI? 16.8) encodes an enterokinase cleavage site
`immediately preceding the polylinker cloning region. Proteins produced as Trx fusions
`using this system can be subsequently released by incubation with enterokinase, leaving
`their authentic amino-terminal sequence. The protocol below describes the use of bovine
`enterokinase in this application.
`
`Additional Materials
`
`For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
`solutions, see APPENDIX 2; for suppliers, see APPENDIX 4.
`
`1 mg/ml thioredoxin fusion protein (UNIT16.8) in 50 mM Tris-Cl (pH 8.0)/1 mM
`CaCl2
`10 ug/ml bovine enterokinase (Biozyme EK-3 grade) in 50 mM Tris-Cl
`(pH 8.0)/1 mM CaCl2
`
`NOTE: Many commercial preparations of enterokinase (bovine or porcine), with the
`exception of the source listed, are extremely impure and tend to be contaminated with,
`among other things, trypsin and chymotrypsin which can extensively degrade the fusion
`protein. It is recommended that only commercial enterokinase of the highest quality be
`used.
`
`1. Perform a pilot experiment to monitor the efficiency of cleavage with various ratios
`of enterokinase to fusion protein. Prepare five reactions:
`
`Reactions I to 4: 20 [11 of 1 mg/ml fusion protein, 1 [L1, 2 [L1, 5 it], and 10 11.1
`of 10 ug/ml bovine enterokinase, and 50 mM Tris-Cl (pH 8.0)/1 mM
`CaCl2, to a total of 30 ul.
`Reaction 5: 20 ul of 1 mg/ml fusion protein and 10 ul of 50 mM Tris-Cl
`(pH 8.0)/1 mM CaCl2 (mock digestion).
`
`Incubate samples 2 16 hr at 37°C.
`
`Current Protocols in Molecular Biology
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`
`Enzymatic and
`Chemical
`_Cleavage_ of
`E15100 Proteins
`
`16.4.10
`
`Supplement 28
`
`

`
`The fusion protein must be (at least) partially purified prior to digestion with entero—
`kinase because the enzyme is inactive in crude bacterial lysates.
`
`2. Stop the reaction by adding 30 ul of 2x SDS sample buffer to each reaction. Boil 10
`min.
`
`For larger-scale applications, the reaction can be stopped by adding p-aminobenza-
`midine (PABA) to 5 mM. PABA is a competitive inhibitor of most intestinal serine
`proteases. It should provide protection from nonspecific proteolysis of the reaction
`products by contaminants in the enzyme preparation before the protein of interest is
`purifiedfiuthen
`
`3. Load 10 pl of each sample onto an SDS-polyacrylamide gel to analyze the extent of
`cleavage. Adjust enterokinase concentration and length of incubation accordingly to
`accomplish complete digestion.
`
`4. Scale up the reaction components linearly to digest a larger amount of fusion protein.
`
`Calcium ions marginally increase the efficiency of cleavage, but their presence
`sometimes promotes fusion protein degradation by stimulating contaminating pro-
`teolytic activities. If degradation of the cleaved fusion protein occurs, omit calcium
`and add 5 mM EDTA to the cleavage reaction to try to eliminate the problem.
`
`CHEMICAL CLEAVAGE OF FUSION PROTEINS USING CYANOGEN
`BROMIDE
`
`BASIC
`PROTOCOL 2
`
`Cyanogen bromide (CNBr) has been used to cleave proteins at methionine residues for
`many years. CNBr has been used industrially for the production of both somatostatin
`(Itakura et al., 1977) and insulin (Chance et al., 1981). The reaction is typically carried
`out at low pH in 70% formic acid, and cleavage occurs at the C-terminal side of methionine
`residues. The protein concentration is relatively unimportant, as the CNBr is in vast excess
`for hydrolysis. The technique is useful only if the protein of interest lacks methionine
`residues. Cleavage with CNBr is usually efficient, but side chain modifications and
`nonspecific cleavages are common upon prolonged incubation at low pH. These prob-
`lems, along with the potential for reduction of intramolecular disulfide bonds during
`treatment with 70% formic acid, can be minimized by replacing the formic acid with 6
`M guanidine-HCI/0.2 M HCl.
`
`CAUTION: Cyanogen bromide is extremely toxic. It should only be used in a properly
`ventilated fume hood. Exercise appropriate caution in its use and disposal.
`
`Materials
`
`For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
`solutions, see APPENDIX 2; for suppliers, see APPENDIX 4.
`
`1 mg/ml fusion protein
`50 mg/ml cyanogen bromide (CNBr)/70% (v/v) formic acid
`70% (v/v) formic acid
`1x SDS sample buffer (UNI? 10.2)
`
`Additional reagents and equipment for SDS—PAGE (UNIT 10.2)
`
`1. Perform a pilot experiment to determine minimum incubation time. Lyophilize two
`50-ul aliquots of fusion protein solution. Resuspend one aliquot in 50 ul of 50 mg/ml
`CNBr/70% formic acid. Resuspend the other in 50 1.11 of 70% formic acid without
`CNBr. Incubate at room temperature.
`
`2. At 0, 8, 24, and 48 hr, remove 21 5-111 aliquot and lyophilize.
`
`Current Protocols in Molecular Biology
`
`Protein
`Expression
`
`16.4.11
`
`Supplement 28
`
`BEQ 1016
`Page 192
`
`

`
`3. Resuspend all aliquots in 20 |.L1 of 1x SDS sample buffer, boil 10 min, and load onto
`an SDS-polyacrylamide gel.
`
`4. Based on analysis of the gel, determine the minimum incubation time necessary to
`completely cleave the protein.
`
`The protocol can be easily scaled up to accommodate larger amounts offusion protein.
`Some proteins are resistant to cleavage with cyanogen bromide. In such cases, or when
`the fitsion protein to be cleaved is insoluble, guanidine-HCl can be added to the
`reaction at afinal concentration of 6 M.
`
`ALTERNATE
`PROTOCOL 4
`
`CHEMICAL CLEAVAGE OF FUSION PROTEINS USING
`HYDROXLAMINE
`
`Hydroxylamine cleaves proteins at Asn—G1y bonds and can be used as a reagent for
`chemical cleavage of fusion proteins. This cleavage site is less common than that for
`cyanogen bromide, and therefore the presence of a susceptible bond in the protein of
`interest is less likely. One disadvantage is that the released carboxy-terminal fusion partner
`will retain a glycine residue at its amino terminus, which is unacceptable in some
`applications. Also, the reaction requires incubation of the fusion protein at alkaline pH,
`which may cause modification of some amino acid side chains. Finally, protein digestions
`by this technique are usually incomplete due to the nature of the cleavage mechanism
`(E.L., unpub. observ.), reducing yield and possibly complicating post-cleavage purifica-
`tion of the desired protein product. However, the technique does have advantages: speed,
`economy, and the ability to perform digestions under denaturing conditions (e.g., 6 M
`guanidine-HCl) for otherwise insoluble fusion proteins.
`
`CAUTION: Hydroxylamine is potentially explosive if mishandled. Be sure to follow all
`precautions indicated by the manufacturer.
`
`Additional Materials
`
`For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
`solutions, see APPENDIX 2; for suppliers, see APPENDIX 4.
`
`1 mg/ml fusion protein in 10 mM Tris-Cl (pH 8.0)/150 mM NaCl
`2x hydroxylamine cleavage solution (see recipe)
`Guanidine-HCl (optional)
`2x SDS sample buffer (UNIT 10.2)
`Boiling water bath
`
`1. Perform a pilot experiment to determine minimum incubation time. Mix 50 ul of 1
`mg/ml fusion protein in 10 mM Tris-Cl (pH 8.0)/150 mM NaCl with 50 pl of 2x
`hydroxylamine cleavage solution in a 1.5-ml microcentrifuge tube. Incubate at 45°C.
`
`Ifthefusion protein is insoluble, guanidine»HCl can be added to the cleavage reaction
`at afinal concentration of6 M. This may also help in cases where a particular Asn-Gly
`bond appears to be resistant to cleavage.
`
`2. At 0, 2, 4, 8, 16, and 24 hr, remove 10-ul aliquots from the cleavage reaction and mix
`with 10 ul of 2x SDS sample buffer. Freeze each tube on dry ice until all time points
`have been collected.
`
`4. Heat samples 10 min in a boiling water bath. Load all samples onto an SDS-poly-
`acrylamide gel to analyze the extent of cleavage.
`
`5. Determine the minimum incubation time necessary for maximum cleavage.
`I_f cleavage after 24 hr is still poor, add guanidine-HCl to 6 M final,
`hydroxylamine concentration to 3 Mfinal, or both.
`
`increase
`
`Cunent Protocols in Molecular Biology
`BEQ 1016
`Page 193
`
`Enzymatic and
`ClEzi1ve::glecta>l'
`Fusion Pmteins
`
`16.4.12
`
`Supplement 28
`
`

`
`CHEMICAL CLEAVAGE OF FUSION PROTEINS BY HYDROLYSIS
`
`AT Low pH
`
`ALTERNATE
`PROTOCOL 5
`
`This method exploits the fact that the Asp-Pro bond is labile at low pH. Hydrolysis of this
`peptide bond occurs at elevated temperatures (37° to 40°C) under acidic conditions (pH
`2.5). Nonspecific cleavages can occur upon prolonged incubation under these conditions,
`and it is necessary to determine empirically the minimum length of time necessary for
`cleavage. Like the other chemical cleavage methods described in this unit, the reaction
`conditions are somewhat harsh and may result in denaturation or modification of the
`protein. On the other hand, this treatment allows insoluble fusion proteins to be cleaved
`by acid hydrolysis of Asp-Pro bonds in the presence of 6 M guanidine-HCl. To use this
`procedure the amino acid sequence of the carboxy-terminal fusion partner should first be
`examined carefully to verify the absence of other Asp-Pro bonds. The released protein
`will retain a proline residue at its amino-terminus.
`
`GST
`This method will potentially cleave any protein containing an Asp-Pro bond.
`fusion vector pGEX1 (UNITI6. 7, and Fig. 16.7.1 therein) contains an Asp-Pro cleavage site
`encoded by the BamHI site of the polylinker cloning region, so fusions at tElS site will
`result in fusion proteins that can be released from GST using this protocol.
`
`Additional Materials
`
`For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock
`solutions, see APPENDIX 2; for suppliers, see APPENDIX 4.
`
`Fusion protein containing an Asp-Pro bond between the component domains
`70% (v/v) formic acid
`13% (v/v) acetic acid
`0.1 M Tris base
`Guanidine-HCl
`
`1. Perform a pilot experiment to determine optimal hydrolysis conditions. Prepare four
`reaction mixtures:
`
`Reaction 1: ~20 ug fusion protein in 70% formic acid
`
`Reaction 2: ~20 ug fusion protein in 70% formic acid/6 M guanidine-HCI
`
`Reaction 3: ~20 ug fusion protein in 13% acetic acid
`
`Reaction 4: ~20 ug fusion protein in 13% acetic acid/6 M guanidine-HCl.
`
`Incubate all samples at 37°C.
`
`2. At 0, 24, 48, and 72 hr, remove a 5-ug aliquot of each reaction mixture and lyophilize
`to dryness.
`
`The 0 time point can serve as the negative control.
`
`3. Resuspend the hydrolyzed protein in 20 ul of 1x SDS sample buffer and neutralize
`by gradual addition of 0.1 M Tris base until the sample turns from yellow to blue.
`Analyze samples on a tricine SDS-polyacrylamide gel for extent of digestion.
`
`4. Choose the mildest condition and shortest incubation time that give the desired extent
`of cleavage. Scale up to larger amounts of fusion protein accordingly.
`
`Ftere is a great deal of variation in the susceptibility ofAsp-Pro bonds to cleavage.
`Some Asp-Pro bonds cleave readily undermild conditions, whereas others are resistant
`to cleavage and require incubation in stronger acid conditions and/or strong denatur-
`ants to attain hydrolysis. Even under strong conditions, some Asp-Pro bonds remain
`uncleaved and others may not be cleaved to completion, i.e., they may be cleaved in
`only a fraction of the proteins. However; to avoid unwanted denaturation or modifica-
`tion of the protein of interest, it is important to determine the mildest conditions that
`give the desired degree of cleavage.
`
`Cl.ll”:‘61'It Protocols in Molecular Biology
`
`Protein
`Expression
`
`16.4.13
`
`Supplement 28
`
`BEQ 1016
`Page 194
`
`

`
`REAGENTS AND SOLUTIONS
`
`Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
`APPENDIX 2; for suppliers, see APPENDIX 4.
`
`Hydroxylamine cleavage solution, 2x
`4 M hydroxylarnine
`0.4 M CHES buffer
`
`Adjust pH to 9.5 with NaOH
`Prepare fresh
`
`Thrombin cleavage buffer
`50 mM Tris-Cl, pH 7.5
`150 mM NaCl
`
`2.5 mM CaCl2
`Store indefinitely at —20°C
`
`COMMENTARY
`
`Background Information
`The use of fusion proteins in Escherichia
`coli for production of proteins from other or-
`ganisms is becoming increasingly popular. The
`principal advantages of fusion proteins include
`high expression levels, ease ofpurification, and
`ease of detection with biochemical or im-
`
`munological reagents. However, early protein
`fusion methods fused the target protein to the
`carboxy-terminus of E. coli B—ga1actosidase;
`such proteins are usually insoluble, have re-
`stricted usefulness (except as antigens for an-
`tibody pE'0ClllCtlOl'l), and require refolding of the
`protein in an attempt to retain biological activ-
`ity. The development of improved fusion pro-
`tein expression systems that are often capable
`of producing properly folded and biologically
`active proteins in E. coli, such as the MBP
`system (wwr 16.6), the GST system (wwr 16.7),
`and the Trx system (UNITI6.8), has increased the
`need for suitable methods of separating the
`protein of interest from the so—called carréer
`protein. Such separation is desirable only after
`the carrier domain has been exploited for its
`particular attributes—e.g, to facilitate specific
`purification or detection, or to enhance stability
`or solubility. Subsequent site-specific cleavage
`of the carrier domain from the correctly folded
`protein of interest then allows evaluation of the
`biological activity of the protein without poten-
`tial interference from a covalently-attached fu-
`sion partner.
`Over the years, many different enzymatic
`and chemical methods for site-specific cleav-
`age of polypeptides have been developed
`(Gross, 1967; Spande et al., 1970; Maroux et
`al., 1971; Landon, 1977; Nagai and Thtzigersen,
`1984; Chang, 1985; Szoka et al., 1986; Smith
`and Johnson, 1988; Gearing, et al., 1989; Ham-
`
`Enzymatic and
`Chemical
`Cleavage of
`Fusion Proteins
`
`16.4.14
`
`Supplement 28
`
`mond et al., 1991). The choice of cleavage
`reagents for those expression vectors obtained
`from molecular biology suppliers that are used
`“off the shelf’ will usually be dictated by the
`recognition sequences that lie at their respec-
`tive fusion junctions. For example, MBP vec-
`tors (UNIT 16.6; Maina et al., 1988) are designed
`for factor Xa cleavage, GST vectors (wvrr 16.7)
`offer a choice of factor Xa, thrombin, or acid
`cleavage, and the Trx vector (UNlTI6.8; LaVa1lie
`et al. , 1993a) utilizes enterolcinase for cleavage.
`However, it is importantto remember that these
`vectors, or any other fusion protein expression
`vector, can be manipulated by the user to in-
`clude junction amino acids that will allow en-
`zymatic or chemical hydrolysis with other
`cleavage reagents.
`
`Critical Parameters and
`
`Troubleshooting
`The cleavage methods described in this unit
`have been chosen based upon their specificity,
`efficiency, and reagent availability. Uitimately,
`the choice of cleavage reagent will depend
`upon many factors. First of all, the primary
`sequence of the protein of interest must be
`scrutinized to identify sequences that will be
`susceptible to the cleavage reagents in ques-
`tion. Because of the highly restricted specific-
`ity in the case of the proteases described in the
`preceding protocols, the occurrence of such
`additional sites is unlikely. However, the use of
`chemical cleavage methods or less specific
`proteases such as trypsin (Lysi or Argi) re-
`quires careful consideration of the composition
`of the protein of interest to avoid unwanted
`fragmentation of the product. Secondly, the
`physical characteristics of the fusion protein
`are an important consideration in choosing an
`
`Current Protocols in Molecular Biology
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`Page 195
`
`

`
`appropriate cleavage method. Fusion proteins
`that are insoluble generally require a chemical
`cleavage method that allows incubation in the
`presence of protein denaturants such as
`guanidine-HCl or urea followed by proper re-
`folding. Conversely, soluble fusion proteins
`should be cleaved under conditions where de-
`
`naturation is minimized to preserve their struc-
`ture and/or biological activity. In addition,
`some cleavage methods require incubation at
`pH extremes, which may result in modifica-
`tions to some of the side chains or aggregation
`and precipitation of the protein and may ulti-
`mately be deleterious to the usefulness of the
`cleaved product.
`When the fusion protein is produced in a
`soluble fashion, enzymatic cleavage protocols
`are preferred for many reasons. These reactions
`are carried out under mild conditions of neu1ral
`
`pH, low ionic strength, and moderate temper-
`ature (25° to 37°C). Such conditions approxi-
`mate physiological environments and typically
`should be least harmful to the integrity of the
`protein. The high degree of specificity exhib-
`ited by these proteases ensures a low probabil-
`ity of unwanted cleavage elsewhere in the pro-
`tein of interest. Finally, the extent of enzymatic
`cleavage typically can be altered by modulat-
`ing parameters such as amount of enzyme,
`subsfzate concentration, and length of incuba-
`tion.
`
`The use of proteases sometimes causes
`problems, however. The most common prob-
`lem is unwanted secondary proteolysis or deg-
`radation of the fusic:-n protein by contaminating
`proteolytic activities in the cleavage reaction.
`Degradation may be caused by E. cali proteases
`that have not been purified away from the
`fusion protein prior to cleavage. Such contam-
`inants can often be alleviated by additional
`purification prior to enzymatic digestion. Al-
`ternatively, the unwanted proteolytic activéty
`may result from a contaminant in the enzyme
`preparation itself. These enzymes are serine
`proteases that have been purified from natural
`sources, so it is probable that they are contam-
`inated with other proteases that copurify in
`trace amounts. For example, even highly puri-
`fied enterokinase from bovine intestine con-
`
`tains Irace amounts of tryptic and chymotryptic
`activity that can cause minor secondary prote-
`olysis of thioredoxin fusion proteins (LaVallie
`et al., 1993a). This degradation can be reduced
`by omitting Ca” ions from the digestion and
`using EDTA to chelate residual Ca”. The best
`solution to this problem, however, is to use a
`recombinant source of enzyme produced in cell
`
`Current Protocols in Molecular Biology
`
`culture; such enzymes are free of contaminat-
`ing proteases found in intesténal preparations
`(LaVallie et al., 1993b and unpub. observ.).
`Another potential problem that has been
`observed with some enzymatic cleavages is
`cleavage at sites other than the anticipated
`peptide bonds. This has been reported for factor
`Xa (Nagai and Thgzsgersen, 1987; Lauritzen et
`al., 1991) and thrombin (Chang, 1985); entero—
`kinase has been observed to cleave at subsites
`
`that resemble the (Asp)4-Lys recognition se-
`quence when the substrate is denatured or oth-
`erwise improperly folded (Light et al., 1980, E.
`LaVallie and L. Racie, unpub. observ.). Almost
`always, this relaxed site specificity can be min-
`imized by decreasing the enzyme/substrate
`ratio and/or the éme of incubation. There are
`
`also more exotic strategies; for example, re-
`versible acylation of the fusion protein has been
`used to eliminate nonspecific cleavage by fac-
`tor Xa (Wearne, 1990).
`Although it is less desirable than enzymatic
`digestion in most circumstances, chemical
`cleavage of fusion proteins is sometimes nec-
`essary and may be advantageous in certain
`applications. This is in spite of the significant
`disadvantages. First, chemical cleavage proce-
`dures almost universally employ harsh condi-
`tions, such as pH extremes or high tempera-
`tures,
`that can denature the fusion protein
`and/or modify amino acid side chains (e.g.,
`deamidation of Asn residues and oxidation of
`
`Met residues). Second, cleavage specifscity
`tends to be limited to single amino acids or, at
`most, dipeptide sequences, greatly decreasing
`the utility of chemical reagents in site—specific
`cleavage of large polypeptide substrates. Even
`this low degree of specificity is not absolute,
`and low levels of side reactions at alternate sites
`
`have been noted for hydroxylamine (Bornstein
`and Balian, 1970; Steinman et al., 1974) and
`cyanogen bromide (Langley and Smith, 1971).
`Even acidic cleavage of Asp—Pro bonds is
`sometimes accompanied by nonspecific pep-
`tide bond hydrolysis when incubation is pro-
`longed (Landon, 1977). Third, using chemical
`cleavage reagents involves the danger of work-
`ing with hazardous compounds such as cyano-
`gen bromide and hydroxylamine. Ex1Ieme care
`must be used in the storage, use, and disposal
`of these reagents. Material Safety Data Sheets
`(MSDSs) should be obtained from the manu-
`facturer for any of these compounds and should
`be read carefully.
`In spite of these shortcomings, however,
`specific chemical hydrolysis of junction pep-
`tide bonds in fusion proteins is preferred for
`
`Protein
`Expression
`
`16.4.15
`
`Supplement 28
`
`BEQ 1016
`Page 196
`
`

`
`some applications. For instance, fusion pro-
`teins that are produced in an insoluble form
`cannot be cleaved enzymatically unless they
`have been solubilized and refolded, but site-
`
`specific cleavage of the protein domains often
`can be accomplished using chemical hydroly-
`sis in the presence of strong chaotropic agents
`such as guanidine-HCl (Landon, 1977; Szoka
`et al., 1986; Villa et al., 1989). Chemical cleav-
`ages are also useful when a soluble fusion pro-
`tein is refractory to cleavage under nondenatur-
`ing conditions using either enzymatic or chem-
`ical cleavage methods. Other advantages of
`chemical cleavage reagents are economy, pu-
`rity, and wide availability.
`
`Anticipated Results
`Enzymatic digestion of fusion proteins is
`sensitive to many different parameters such as
`temperature, pH, ionic strength, buffer compo-
`sition, substaate concentration, enzyme con-
`centration, and length of incubation. Optimum
`parameters must be deterznined empirically for
`each fusion protein. However, if the fusion
`protein is of adequate purity and the cleavage
`site is accessible, in most cases the standard
`conditions described in the protocols will give
`satisfactory cleavage.
`Chemical cleavages are much more forgiv-
`ing of small variations in reaction conditions,
`and cleavage should be attainable using the
`reaction conditions given. However, the extent
`of protein modifications caused by these gen-
`eral reaction conditions may often be signifi-
`cant to the ultimate usefulness of the cleaved
`
`protein. Reaction parameters may be adjusted
`as described to minimize any modifications or
`secondary cleavages that may occur.
`With any of these methods, complete diges-
`tion of the fusion protein is often difficult to
`attain, and cleavage efficiencies of 70% to 80%
`should be considered satisfactory.
`
`Time Considerations
`
`Fusion protein cleavage can be accom-
`plished in hours or days, depending upon the
`reagents used and the reaction conditions. For
`factor Xa, thrombin, and enterokinase, the en-
`zyme—to-substrate ratio and substrate concen-
`tration are often chosen so the digestions are
`usually complete in 8 to 24 hr. By contrast,
`chemical cleavages are often highly variable in
`the amount oftime necessary for total cleavage.
`Overnight digestion under standard conditions
`is common for cyanogen bromide and acid
`cleavages, whereas hydroxylamine cleavages
`are often complete in 2 to 4 hr.
`
`Literature Cited
`Bomstein, P. and Balian, G. 1970. The specific
`nonenzymatic cleavage of bovine ribonuclease
`with hydroxylamine. J. Biol. Chem. 245:4854-
`4856.
`
`Bomstein, P. and Balian, G. 1977. Cleavage at Asn-
`Gly bonds with hydroxylarnine. Methods En-
`zymol. 47: 132-145.
`Chance, R.E., Hoffman, J.A., Kroeff, E.P., Johnson,
`M.G., Schirmer, E.W., Bromer, W.W., Ross,
`M.J., and Wetzel, R. 1981. The production of
`human insulin using recombinant DNA technol-
`ogy and a new chain combination procedure. In
`Peptides: Synthesis-Structure-Function (D.H.
`Rich and E. Gross, eds.), pp. 721-728. Pierce
`Chemical, Rockford, Ill.
`
`Chang, J.-Y. 1985. Thrombin specificity. Eur. J.
`Biochem. 15l:217-224.
`
`Gearing, D.P., Nicola, N.A., Metcalf, D., Foote, S.,
`Willson, T.A., Gough, N.M., and Williams, R.L.
`1989. Production of leukemia factor in Es-
`cherichia coli by a novel procedure and its use
`in maintaining embryonic stem cells in culture.
`Bio/Technology 7: 1 157-1161.
`
`Gross, E. 1967. The cyanogen bromide reaction.
`Methods Enzymol. 1 1 1238-255.
`Hammond, P.M., Atkinson, T., Sherwood, R.F., and
`Scawen, M.D. 1991. Manufacturing new-gener-
`ation proteins. Part I: The technology. BioPharm.
`4: 16-23.
`
`ltakura, K., Hirose, T., Crea, R., Riggs, A.D., Heyne-
`ker, H.L., Bolivar, F., and Boyer, H.W. 1977.
`Expression in Escherichia coli of a chemically
`synthesized gene for the hormone somatostatin.
`Science l98:1056-1063.
`
`Langley, T]. and Smith, E.L. 1971. Sequence of
`bovine liver glutamate dehydrogenase. IV. Cy-
`anogen bromide peptides. J. Biol. Chem.
`2746:3789-3801.
`
`Landon, M. 1977. Cleavage at aspartyl-prolyl
`bonds. Methods Enzymol. 47(E): 145-149.
`Lauritzen, C., Tiichsen, E., Hansen, P.E., and
`Skovgaard, O. 1991. BPTI and N-terminal ex-
`tended analogues generated by Factor Xa cleav-
`age and cathepsin C trimming of a fusion protein
`expressed in Escherichia coli. Pnot. Expr. Pursf
`2: 372-378.
`
`LaVa1lie, E.R., DiBlasio, E.A., Kovacic, S., Grant,
`K.L., Schendel, RE, and McCoy, J .M. 1993a. A
`thioredoxin gene fusion system that circumvents
`inclusion body formation in the E. coli cyto-
`plasm. Bio/Technalogy 11:187-193.
`LaVallie, E.R., Rehemtulla, A., Racie, L.A.,
`DiBlasio, E.A., Ferenz, C., Grant, K.L., Light,
`A., and McCoy, J.M. 1993b. Cloning and func-
`tional expression of a cDNA encoding the
`catalytic subunit of bovine enterokinase. J. Biol.
`Chem. 268:23311-23317.
`
`Light, A., Savithri, H. S., and Liepnieks, J.J. 1980.
`Specificity of bovine enterokinase toward pro-
`tein substrates. Anal. Biochem. 106: 199-206.
`
`Current Protocols in Molecular Biology
`BEQ 1016
`Page 197
`
`Enzymatic and
`Chemical
`Cleavage of
`Fusion Proteins
`
`16.4.16
`
`Supplement 28
`
`

`
`Maina, C.V., Riggs, P.D., Grandea, A.G., Slatko,
`B.E., Moran, L.S., Tagliamonte, J.A., McRey—
`nolds, L.A., and Guan, C. 1988. An Escherichia
`coli vector to express and purify foreigr: proteins
`by fusion to and separation from maltose-bind-
`ing protein. Gene 74:365-373.
`Maroux, S., Baratti, J., and Desnuelle, P. 1971.
`Purification and specificity of porcine enteroki-
`nase. .1. Biol. Chem. 246:5031-5039.
`
`Nagai, K. and Thogersen, H. C. 1984. Generation of
`B-globin by sequence-specific proteoiysis of a
`hybrid protein in Escherichia coli. Nature
`309:810-812.
`
`Nagai, K. and Thogersen, H. C. 1987. Synthesis and
`sequence-specific proteolysis of hybrid proteins
`produced in Escherichia coli. Methods EnzymoL
`153:461—481.
`
`Smith, D.B. and Johnson, K.S. 1988. Single-step
`purification of polypeptides expressed in Es-
`cherichia coli as fusions with glutathione S-
`transferase. Gene 67:31-40.
`
`Spande, T.F., Witkop, 13., Degani, Y., and Pat-
`chomik, A. 1970. Selective cleavage and modi—
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`Chem. 24:97—260.
`
`Steinman, H.M., Naik, V.R., Abemethy, J.L., and
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`dismutase: Complete amino acid sequence. J.
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`
`Szoka, P.R., Schreiber, A.B., Chan, H., and Murthy,
`J . 1986. A general method for retrieving compo—
`nents of a genetically engineered fusion protein.
`DNA 5:11-20.
`
`Villa, S., DeFazio, G., and Canosi, U. 1989. Cyano-
`gen bromide cleavage at methionine residues of
`polypeptides containing disulfide bonds. Anal.
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`
`Wearne, S.J. 1990. Factor Xa cleavage of fusion
`proteins: Elimination of nonspecific cleavage by
`reversible acylation. FEBS Lett. 263:23—26.
`
`Contributed by Edward R. LaVallie
`and John M. McCoy
`Genetics Institute
`
`Cambridge, Massachusetts
`
`Donald B. Smith (throirebin cleavage)
`University of Edinburgh
`Edinburgh, Scotland
`
`Paul Riggs (denaturing fusion proteins)
`New England Biolabs
`Beverly, Massachusetts
`
`Current Protocols in Molecular Biology
`
`Protein
`Expression
`
`16.4.17
`
`Supplement 28
`
`BEQ 1016
`Page 198
`
`

`
`UNIT 16.5 Expression and Purification of lacZ and
`trpE