GE Healthcare
`
`Recombinant Protein
`Purification Handbook
`
`Principles and Methods
`
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`1 of 306
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`Handbooks
`from GE Healthcare
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`... _ --
`2:. - - . . : : : i .
`
`·-r- L_.::2---
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`:=-;/ •::•:-:-:
`
`Protein Purification
`Handbook
`18-1132-29
`
`Gel Filtration
`Principles and Methods
`18-1022-18
`
`Affinity Chromatography
`Principles and Methods
`18-1022-29
`
`Antibody Purification
`Handbook
`18-1037-46
`
`Cell Separation Media
`Methodology and Applications
`18-1115-69
`
`Ion Exchange Chromatography and Chromatofocusing
`Principles and Methods
`11-0004-21
`
`Purifying Challenging Proteins
`Principles and Methods
`28-9095-31
`
`GST Gene Fusion System
`Handbook
`18-1157-58
`
`Hydrophobic Interaction and Reversed Phase
`Chromatography
`Principles and Methods
`11-0012-69
`
`2-D Electrophoresis using immobilized pH gradients
`Principles and Methods
`80-6429-60
`
`Microcarrier Cell Culture
`Principles and Methods
`18-1140-62
`
`Recombinant Protein Purification Handbook
`Principles and Methods
`18-1142-75
`
`Isolation of mononuclear cells
`Methodology and Applications
`18-1152-69
`
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`Recombinant Protein
`Purification Handbook
`
`Principles and Methods
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`Content
`
`Introduction .............................................................................................................................................. 5
`Chapter 1
`Expression and sample preparation .................................................................................................... 9
`Components of the expression system ....................................................................................................................... 9
`Sample preparation ............................................................................................................................................................14
`Chapter 2
`Manual and automated purification .................................................................................................. 21
`Tagged recombinant proteins for simple purification ........................................................................................21
`Manual purification techniques .....................................................................................................................................21
`Automated purification using ÄKTAdesign chromatography systems .......................................................22
`Chapter 3
`Purification of histidine-tagged recombinant proteins .................................................................. 25
`Expression ................................................................................................................................................................................25
`Purification overview ..........................................................................................................................................................25
`Purification using precharged media .........................................................................................................................31
`Purification using Ni Sepharose High Performance .............................................................................................33
`Purification using Ni Sepharose 6 Fast Flow ...........................................................................................................37
`High-throughput screening using His MultiTrap HP and His MultiTrap FF 96-well filter plates ......42
`Minipreps using His SpinTrap and His SpinTrap Kit ..............................................................................................47
`Purification using HisTrap HP and HisTrap FF ........................................................................................................50
`Purification using HisTrap FF with ÄKTAprime plus. .............................................................................................56
`Purification from unclarified cell lysate using HisTrap FF crude ....................................................................59
`Manual purification using HisTrap FF crude Kit with a syringe ......................................................................66
`Gravity-flow purification using His GraviTrap and His GraviTrap Kit ...........................................................72
`Scale-up purification using HisPrep FF 16/10 .........................................................................................................76
`Purification using uncharged media ...........................................................................................................................78
`Purification using IMAC Sepharose High Performance ......................................................................................80
`Purification using IMAC Sepharose 6 Fast Flow .....................................................................................................83
`Purification using HiTrap IMAC HP and HiTrap IMAC FF columns .................................................................86
`Preparative purification using HiPrep IMAC FF 16/10 column ........................................................................90
`Detection of histidine-tagged proteins ......................................................................................................................94
`Tag removal by enzymatic cleavage ..........................................................................................................................97
`Troubleshooting ....................................................................................................................................................................99
`Chapter 4
`Optimizing purification of histidine-tagged proteins ...................................................................103
`Optimizing using imidazole ...........................................................................................................................................103
`Optimizing using different metal ions ......................................................................................................................106
`Optimizing using multistep purifications ................................................................................................................109
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`Chapter 5
`Purification of GST-tagged recombinant proteins ........................................................................111
`Expression .............................................................................................................................................................................118
`Purification ...........................................................................................................................................................................120
`General considerations for purification of GST-tagged proteins ................................................................121
`Selecting equipment for purification ........................................................................................................................122
`Purification using Glutathione Sepharose High Performance,
`Glutathione Sepharose 4 Fast Flow, and Glutathione Sepharose 4B .......................................................123
`High-throughput screening using GST MultiTrap FF
`and GST MultiTrap 4B 96-well filter plates.............................................................................................................129
`Minipreps using the GST SpinTrap Purification Module ...................................................................................133
`Gravity-flow purification using the Bulk or RediPack GST Purification Modules .................................135
`Purification using GSTrap HP, GSTrap FF, and GSTrap 4B columns ...........................................................138
`Purification of a GST-tagged protein using GSTrap FF 1 ml with ÄKTAprime plus .............................141
`Preparative purification using GSTPrep FF 16/10 column .............................................................................144
`Troubleshooting of purification methods ..............................................................................................................150
`Detection of GST-tagged proteins .............................................................................................................................154
`Troubleshooting of detection methods ...................................................................................................................163
`Removal of GST tag by enzymatic cleavage ........................................................................................................165
`Troubleshooting of cleavage methods ...................................................................................................................179
`Chapter 6
`Purification of MBP-tagged recombinant proteins .......................................................................181
`Purification using MBPTrap HP columns ................................................................................................................186
`Troubleshooting .................................................................................................................................................................191
`Chapter 7
`Purification of Strep-tag II recombinant proteins .........................................................................193
`Purification using StrepTactin Sepharose High Performance ......................................................................193
`Purification using StrepTrap HP 1 ml and 5 ml .......................................................................................................96
`Troubleshooting .................................................................................................................................................................203
`Chapter 8
`Simple purification of other recombinant or native proteins .....................................................205
`Ready-to-use affinity purification columns ..........................................................................................................205
`Making a specific purification column ....................................................................................................................207
`Purification ...........................................................................................................................................................................209
`Chapter 9
`Multistep purification of tagged and untagged recombinant proteins ....................................211
`Selection and combination of purification techniques....................................................................................212
`Chapter 10
`Handling inclusion bodies ..................................................................................................................221
`Optimizing for soluble expression .............................................................................................................................221
`Refolding of solubilized recombinant proteins ....................................................................................................223
`Troubleshooting .................................................................................................................................................................228
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`Chapter 11
`Desalting, buffer exchange, and concentration ............................................................................229
`General considerations ..................................................................................................................................................233
`Small-scale desalting and buffer exchange with PD desalting columns ................................................234
`HiTrap Desalting columns .............................................................................................................................................243
`Automated desalting with HiTrap Desalting columns on ÄKTAprime plus ............................................245
`Scaling up desalting from HiTrap to HiPrep Desalting.....................................................................................246
`Automated buffer exchange on HiPrep 26/10 Desalting with ÄKTAprime plus...................................247
`Protein sample concentration .....................................................................................................................................249
`Appendix 1
`Characteristics of Ni Sepharose and uncharged IMAC Sepharose products ........................................251
` Ni Sepharose products ...............................................................................................................................................251
` Uncharged IMAC Sepharose products ...............................................................................................................257
`Appendix 2
`Characteristics of Glutathione Sepharose products .........................................................................................263
`Appendix 3
`Characteristics of Dextrin Sepharose High Performance products ..........................................................267
`Appendix 4
`Characteristics of StrepTactin Sepharose High Performance products ..................................................269
`Appendix 5
`Precipitation and resolubilization ..............................................................................................................................273
`Appendix 6
`Column packing and preparation .............................................................................................................................277
`Appendix 7
`Conversion data .................................................................................................................................................................280
`Appendix 8
`Converting from linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa ......................281
`Appendix 9
`GST vectors...........................................................................................................................................................................282
`Control regions for pGEX vectors ...............................................................................................................................283
`Appendix 10
`Amino acids table ..............................................................................................................................................................284
`Appendix 11
`Principles and standard conditions for different purification techniques ..............................................287
`Appendix 12
`Tables for Vivaspin sample concentrators ............................................................................................................287
`Product index ........................................................................................................................................295
`Related literature ................................................................................................................................297
`Ordering information ..........................................................................................................................298
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`Introduction
`
`This handbook is intended for those interested in the expression and purification of recombinant
`proteins. The use of recombinant proteins has increased greatly in recent years, as has the
`wealth of techniques and products used for their expression and purification. The advantages
`of using a protein/peptide tag fused to the recombinant protein to facilitate its purification and
`detection is now widely recognized. In some cases, tags may improve the stability and solubility
`of recombinant proteins.
`The reader will be introduced to the initial considerations to be made when deciding upon
`host, vector, and use of a tagged or untagged protein. General guidelines for successful protein
`expression are also included. Advice is given on harvesting and extraction, handling of inclusion
`bodies, tag removal, and removal of unwanted salts and small molecules.
`Purification of recombinant proteins can be performed manually or by using a chromatography
`system. The system can be operated manually or it can be automated to save time and effort.
`The purification can be performed on many scales, in columns of various sizes. Columns can
`be purchased prepacked with a chromatographic medium, or empty columns can be packed
`manually. Purification can also be performed in batch, with gravity flow or centrifugation, in
`SpinTrap™ columns using centrifugation, or in a 96-well plate format using MultiTrap™ products.
`Proteins are purified using chromatography techniques that separate them according to
`differences in their specific properties, as shown in Figure 1. Tags enable recombinant proteins
`to be purified by affinity chromatography, which is designed to capture the tagged recombinant
`protein based on biorecognition of the tag. Thus, several different recombinant proteins can be
`purified by the same affinity technique if they all have the same tag. In the same way, tags also
`allow the use of a common detection protocol for different recombinant proteins. Consequently,
`tagged proteins are simple and convenient to work with and, for many applications, a single
`purification step, using a commercially available chromatography column, is sufficient. This
`is clearly demonstrated in the specific chapters on the expression, purification, and detection
`of recombinant proteins fused with the commonly used histidine, glutathione S-transferase
`(GST), maltose binding protein (MBP), or Strep-tag™ II tags. A scheme for the general purification
`of histidine-tagged proteins is given in Figure 2. In addition, suggestions for the successful
`purification of untagged recombinant proteins by a single affinity chromatography step are
`also given in this handbook. When a higher degree of purity is required for either tagged or
`untagged recombinant proteins, a multistep purification will be necessary. This can become a
`straightforward task by choosing the right combination of purification techniques.
`
`Gel filtration
`
`Hydrophobic interaction
`
`Ion exchange
`
`Affinity
`
`Reversed phase
`
`Fig 1. Separation principles in chromatographic purification.
`
`In summary, this handbook aims to help the reader achieve a protein preparation that contains
`the recombinant protein of interest in the desired quantity and quality required for their particular
`needs. The quality of the recombinant protein can be reflected in its folding and biological activity.
`
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`General purification of histidine-tagged proteins
`
`Native
`conditions
`
`Binding buffer
`(including 20 to
`40 mM imidazole)
`
`D
`D
`D
`
`D
`D
`
`D
`
`Cell lysis
`
`✓
`
`Binding to
`affinity media
`
`Binding buffer
`(including 20 to
`40 mM imidazole)
`
`DD . .
`Wash
`D
`
`Denaturing
`conditions
`
`
`Binding bufferBinding buffer
`(including 20 to
`(including 20 to
`40 mM imidazole
`40 mM imidazole
`and 8 M urea
`and 8 M urea
`or 6 M guanidine
`or 6 M guanidine
`hydrochloride)
`hydrochloride)
`
`Binding buffer
`(including 20 to
`40 mM imidazole
`and 8 M urea
`or 6 M guanidine
`hydrochloride)
`
`Elution buffer: Binding buffer
`with a higher concentration
`of imidazole
`
`Elute
`
`On-column
`refolding
`
`Elution buffer: Binding buffer
`with a higher concentration
`of imidazole
`
`Purified
`tagged
`protein
`
`Elute
`
`Purified
` denatured
`tagged
`protein
`
`D
`
`Purified
`tagged
`protein
`
`Off-column refolding
`
`Purified
`tagged
`protein
`
`tagged protein
`
`cell protein
`
`denatured tagged protein
`
`Fig 2. General purification workflow for histidine-tagged proteins (assumes use of Ni2+-charged affinity media, but other
`metal-ion-charged media follow a similar workflow).
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`Common acronyms and abbreviations
`A280
`UV absorbance at specified wavelength (in this example, 280 nanometers)
`AC
`affinity chromatography
`BCA
`bicinchoninic acid
`CDNB
`1-chloro-2,4-dinitrobenzene
`CF
`chromatofocusing
`CIPP
`Capture, Intermediate Purification, and Polishing
`CV
`column volume
`DAB
`3,3’-diaminobenzidine
`DNase
`deoxyribonuclease
`ELISA
`enzyme-linked immunosorbent assay
`FF
`Fast Flow
`Gua-HCl
`guanidine-HCl
`GF
`gel filtration
`GST
`glutathione S-transferase
`HIC
`hydrophobic interaction chromatography
`HMW
`high molecular weight
`HP
`High Performance
`HRP
`horseradish peroxidase
`IEX
`ion exchange chromatography
`IMAC
`immobilized metal ion affinity chromatography
`isopropyl β-D-thiogalactoside
`IPTG
`LMW
`low molecular weight
`MBP
`maltose binding protein
`MPa
`megaPascal
`Mr
`relative molecular weight
`N/m
`column efficiency expressed as theoretical plates per meter
`PBS
`phosphate buffered saline
`pI
`isoelectric point, the pH at which a protein has zero net surface charge
`psi
`pounds per square inch
`PMSF
`phenylmethylsulfonyl fluoride
`PVDF
`polyvinylidene fluoride
`r
`recombinant, as in rGST and rBCA
`RNase
`ribonuclease
`RPC
`reverse phase chromatography
`SDS
`sodium dodecyl sulfate
`SDS-PAGE
`sodium dodecyl sulfate polyacrylamide gel electrophoresis
`TCEP
`Tris(2-carboxyethyl)phosphine hydrochloride
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`Symbols
`
` this symbol indicates general advice to improve procedures or recommend action under
`specific situations.
`
` this symbol denotes mandatory advice and gives a warning when special care should
`be taken.
`
`
`
`
`
` highlights chemicals, buffers and equipment.
`
` outline of experimental protocol.
`
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`Chapter 1
`Expression and sample preparation
`Components of the expression system
`A protein expression system includes, among other things, a vector with an appropriate promoter
`and other regulatory sequences, along with the gene encoding the recombinant protein of
`interest. Vectors are available commercially for the expression of recombinant proteins either
`fused to a tag or untagged. Such expression vectors are designed with control regions to suit
`the specific host (for example, E. coli versus mammalian cells) and type of expression needed.
`The presence of resistance markers makes selection of the correct clones more straightforward.
`Expression of the recombinant protein can be constitutive or regulated, or it can be at a high or
`low level, depending on the specific requirements. The choice of vector is important because
`it affects so many of the processes that follow the cloning steps including expression, protein
`processing, and purification. The completed vector construct is used in a prokaryotic or eukaryotic
`organism, tissue, or cell line to produce the recombinant protein that may be of academic and/or
`industrial importance. The recombinant protein may then need to be detected, quantitated, and/or
`purified. Selection of a suitable expression system depends on the desired scale of production,
`the time and resources available, and the intended use of the recombinant protein. Several
`alternative systems for expression may be suitable.
`Choice of host
`Many host systems are available including bacteria, yeast, plants, filamentous fungi, insect or
`mammalian cells grown in culture, and transgenic animals or plants. Each host system has its
`own advantages and disadvantages, and it is important to consider these before final selection
`of host.
`The choice of host affects not only the expression of the protein but also the way in which the
`product can be subsequently purified. In order to decide which host is most suitable, the amount
`and the degree of purity of the product, as well as its biological integrity and potential toxicity,
`should be considered. For example, bacterial expression systems are not suitable if post-
`translational modification is required to produce a fully functional recombinant product. Table
`1.1 summarizes features of several expression systems.
`
`Insect cells
`–
`+
`+
`–
`+
`
`Mammalian cells
`–
`+
`+
`–
`+
`
`Table 1.1. Features of several types of expression systems.
`Processing
`Bacteria
`Yeast
`Inclusion bodies
`+/-
`(+)/-
`Secretion
`+/–
`+1
`Glycosylation
`–
`+2
`Proteolytic cleavage
`+/–
`+/–
`Other post-translational
`–
`+3
`modifications
`+ = Yes
`– = No
`1 Constructs are often prepared to allow secretion of the protein. This eliminates the need for cell lysis, which requires more
`powerful methods for yeast than for E. coli.
`2 Yeast give more extensive glycosylation than insect cells and mammalian cells; this is a drawback of heterologous
`expression in yeast.
`3 Yeast lack some functions of post-translational modifications that exist in higher eukaryotes.
`The location of product within the host will affect the choice of methods for isolation and
`purification of the product. For example, in addition to expressing the protein cytoplasmically, a
`bacterial host may secrete the protein into the growth medium, transport it to the periplasmic
`space, or store it as insoluble inclusion bodies within the cytoplasm (Fig 1.1). Expression in
`different parts of the cell will lead to varying amounts of cellular (contaminant) proteins that will
`need to be removed to obtain a pure target protein.
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`The main focus of this handbook is purification of soluble proteins from bacterial sources, as
`these are the most common systems. Purification of proteins expressed as inclusion bodies is
`also discussed (see Chapter 10).
`
`70 Å
`
`70 Å
`
`210 Å
`
`70 Å
`
`Culture medium
`~10 proteins
`
`Periplasm
`~100 proteins
`
`Cytoplasm
`~2000 proteins
`
`Lipopolysaccharide
`
`Outer membrane
`
`Peptidoglycan
`
`Inner membrane
`
`Fig 1.1. Schematic cross-section of the cell wall and typical number of protein species in E. coli.
`Choice of vector
`The choice of vector family is largely governed by the host. Once the host has been selected, many
`different vectors are available for consideration, from simple expression vectors to those that
`contain specialized sequences needed to secrete the recombinant proteins. In order to clone the
`gene of interest, all engineered vectors have a selection of unique restriction sites downstream of
`a transcription promoter sequence. Recent developments in cloning technology provide increased
`flexibility in the choice of host and vector systems, including options allowing the DNA sequence of
`interest to be inserted into multiple types of expression vectors.
`The expression of a recombinant protein fused to a tag of known size and biological function can
`greatly simplify subsequent purification and detection (for expression method development and
`purification). In some cases, the protein yield can also be increased. Table 1.2 reviews some of
`the features of tagged protein expression, purification, and detection that may influence the final
`choice of vector.
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`Table 1.2. Advantages and disadvantages of tagged versus untagged protein expression.
`
`Disadvantages
`
`Tag may interfere with protein structure and affect
`folding and biological activity.
`If tag needs to be removed, cleavage may not
`always be achieved at 100%, and sometimes
`amino acids may be left1.
`
`Advantages
`Tagged proteins
`Simple purification is possible using affinity
`chromatography. Generic two-step purification
`protocols can often be set up for lab-scale protein
`production platforms.
`Detection of the tag instead of the target protein
`moiety allows for a generic detection method in, e.g.,
`protein production platforms for structural biology.
`Solubility and stability can be improved.
`Targeting information can be incorporated into a tag.
`A marker for expression is provided.
`Some tags allow strong binding to
`chromatography media in the presence of
`denaturants, making on-column refolding possible.
`Untagged proteins
`
`Tag removal is not necessary.
`
`Purification and detection not as simple.
`
`Problems with solubility and stability may be
`difficult to overcome, reducing potential yield.
`1 The effectiveness of proteases used for cleavage may be decreased by substances, for example, detergents, in the protein
`preparation or by inappropriate conditions.
`
`Choice of tag
`There are several affinity tags that can be used to simplify protein purification. The choice of
`tag may depend on many different factors. The most common tag, the histidine tag, is often a
`(histidine)6, but other polyhistidine tags consisting of between four and 10 histidine residues have
`been used. The latter provides for the strongest affinity for the chromatography medium. Other
`important tags are the GST and MBP tags, both of which are proteins, and Strep-tag II, which is a
`peptide optimized for chromatography on Strep-Tactin™ based chromatography media.
`Table 1.3 on the following page highlights some key features of these tags.
`
`GE Healthcare provides a variety of solutions for purification of histidine-, GST-, MBP- and
`Strep-tag II-tagged proteins. Chapters 3, 5, 6, and 7, respectively, discuss these solutions in
`detail. GE Healthcare provides purification solutions for other tagged proteins as well, including
`the calmodulin-binding peptide, the protein A tag, biotinylated peptide tags, and immunoglobulin
`Fc domain tags. Recombinant proteins fused to the calmodulin-binding peptide can be purified
`by Calmodulin Sepharose™ 4B. Protein A-tagged proteins can be purified using IgG Sepharose
`Fast Flow. Recombinant proteins with a biotinylated peptide tag can be purified using HiTrap™
`Streptavidin HP columns or by using Streptavidin Sepharose High Performance. Immunoglobulin
`Fc domain-tagged proteins can be purified with different Protein A Sepharose or Protein G Sepharose
`chromatography media.
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` Table 1.3. Characteristics of affinity tags.
`Tag-specific characteristics
`Histidine tag
`
`Compatible expression systems
`
`Can be used in any expression system.
`
`GST tag
`Can be used in any expression system.
`
`Metabolic burden to host
`
`Low metabolic burden to expression host. High metabolic burden to expression host.
`
`Yield after purification
`
`Purity in a single step
`
`Purification procedure gives high yields.
`
`Purification procedure gives high yields.
`
`Allows relatively high purity in a single
`purification step. Optimization of washing
`and elution conditions is recommended
`when extra high purity is needed in a
`single step.
`
`Allows extremely high purity in a single
`purification step.
`
`Effect on solubility of expressed protein
`
`Does not enhance solubility.
`
`Selection of purification products
`available for any scale.
`
`Small tag may not need to be removed
`(e.g., tag is weakly immunogenic so target
`protein can be used directly as an antigen
`for immunization). Site-specific proteases1
`enable cleavage of tag if required. TEV
`protease is often used to cleave off
`histidine tags. Note: Enterokinase sites
`that enable tag cleavage without leaving
`behind extra amino acids are preferable.
`
`Histidine tag is easily detected using anti-
`His-based immunoassay.
`
`Simple purification. Note: Imidazole may
`cause precipitation in rare cases. Buffer
`exchange to remove imidazole may be
`necessary (see Chapter 11).
`
`
`Can be used for dual tagging to
`increase purity and to secure full-length
`polypeptides if the tags are placed at the
`N- and C-terminals.
`Dual tagging in combination with
`Strep-tag II minimizes effects on the
`target protein due to the small size of
`both tags.
`
`Purification can be performed under
`denaturing conditions if required. Allows
`on-column refolding.
`
`Mild elution conditions.
`
`Very mild elution conditions.
`
`Purification products for different scales
`
`Affinity tag removal
`
`Tag detection
`
`Ease of purification
`
`
`Elution conditions
`
`Suitability for dual tagging
`
`Suitability for purification under
`denaturing conditions
`
`Effect on protease action
`
`No effect on protease action.
`
`Effect on folding
`
`Minimal effect on folding.
`
`Effect on structure and function of
`fusion partner
`
`Small tag is less likely to interfere with
`structure and function of fusion partner.
`
`Effect on crystallization
`
`Suitability for purification of protein
`complexes
`
`Suitability for purification of proteins
`containing metal ions
`
`12 18-1142-75 AD
`
`Less risk of effects on crystallization than
`for large tags. May allow crysta