GE Healthcare
`
`Purifying
`Challenging Proteins
`Principles and Methods
`
`•
`
`1 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Handbooks
`from GE Healthcare
`
`~ _ ...
`~-~-
`
`I
`.. 1
`.A
`
`I~
`
` . ... :.-••
`
`.. -
`~
`
`- - - - - ,
`... ~ :::----
`::!.J ~
`, ~-
`
`---
`
`0
`I O
`~ - - - l
`
`0
`----1 0
`
`M,cr0tarno,
`Cel1cu1mre
`
`----
`
`GST Gene Fusion System
`Handbook
`18-1157-58
`
`Affinity Chromatography
`Principles and Methods
`18-1022-29
`
`Antibody Purification
`Handbook
`18-1037-46
`
`Percoll
`Methodology and Applications
`18-1115-69
`
`Ion Exchange Chromatography
`& Chromatofocusing
`Principles and Methods
`11-0004-21
`
`Purifying
`Challenging Proteins
`Principles and Methods
`28-9095-31
`
`Gel Filtration
`Principles and Methods
`18-1022-18
`
`Recombinant Protein
`Purification Handbook
`Principles and Methods
`18-1142-75
`
`Protein Purification
`Handbook
`18-1132-29
`
`Hydrophobic Interaction and
`Reversed Phase Chromatography
`Principles and Methods
`11-0012-69
`
`2-D Electrophoresis
`using immobilized pH gradients
`Principles and Methods
`eo-6429-Go
`
`Microcarrier Cell Culture
`Principles and Methods
`18-1140-62
`
`2 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Purifying
`Challenging Proteins
`Principles and Methods
`
`3 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Contents
`
`Introduction ............................................................................................................................................................... 5
`Challenging proteins ......................................................................................................................................................... 5
`
`Outline .................................................................................................................................................................................... 5
`
`Common acronyms and abbreviations .................................................................................................................. 6
`
`Symbols ................................................................................................................................................................................. ?
`
`Chapter 1
`Membrane proteins ........................................................................................................................................................... 9
`
`Introduction .......................................................................................................................................................................... 9
`
`Classification of membrane proteins ................................................................................................................... 10
`
`Purification of integral membrane proteins for structural and fu nctional studies ......................... 11
`
`Starting material ............................................................................................................................................................. 12
`
`Small-scale expression screening of histidine-taggec membrane proteins
`from E. coli lysates ......................................................................................................................................................... 15
`
`Cell harvest ...................................................................................................................................................................... 17
`
`Cell disruption and membrane preparation ...................................................................................................... 18
`
`Solubilization .................................................................................................................................................................... 20
`
`Purification ........................................................................................................................................................................ 29
`
`Purification of non-tagged membrane proteins ............................................................................................. 38
`
`Purity and homogeneity check ............................................................................................................................... 40
`
`Conditioning ...................................................................................................................................................................... 43
`
`Proteomic analysis of membrane proteins ........................................................................................................ 45
`
`References ......................................................................................................................................................................... 49
`
`Chapter 2
`Multiprotein complexes ............................................................................................................................................... 51
`
`Introduction ....................................................................................................................................................................... 51
`
`Pull-down assays ........................................................................................................................................................... 53
`
`Isolation of native complexes ................................................................................................................................... 61
`
`Isolation of recombinant protein complexes .................................................................................................... 63
`
`References ......................................................................................................................................................................... 67
`
`Chapter 3
`Inclusion bodies .................................................................................................................... , ............................. , ........... 69
`
`Optimizing for soluble expression .......................................................................................................................... 69
`
`Strategies for handling inclusion bodies ............................................................................................................. 70
`
`Isolation of inclusion bodies ...................................................................................................................................... 71
`
`Solubilization .................................................................................................................................................................... 72
`
`4 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Refolding ........................................................................................................................................................................... 72
`
`Refolding using gel fi ltration ..................................................................................................................................... 74
`
`Analysis of refolding ..................................................................................................................................................... 82
`
`References ......................................................................................................................................................................... 82
`
`Appendix 1
`Principles and standard conditions for different purification techniques .......................... ·-··············83
`
`Affinity chromatography (ACl ................................................................................................................................... 83
`
`Ion exchange chromatography (IEXI .................................................................................................................... 84
`
`Hydrophobic interaction chromatography (HIC) ............................................................................................ 86
`
`Gel filtration (GF) chromatography ........................................................................................................................ 87
`
`Reversed phase chromatography [RPC) ............................................................................................................. 88
`
`Appendix 2
`Manual and automated purificotion ............. ........................................................................................................ 89
`
`Togged recombinant proteins for simple purification .................................................................................. 89
`
`Manual purification techniques ............................................................................................................................... 89
`
`Automated purification using AKTAdesign chromatography systems ................................................ 90
`
`Appendix 3
`Column pocking and preparation ......................................................................................................................... 93
`
`Column selection ............................................................................................................................................................ 95
`
`Appendix 4
`Conversion data: proteins, column pressures .......................................................................................................... 96
`
`Column pressures .......................................................................................................................................................... 96
`
`Appendix 5 ................................................................................................................................................................... 97
`Converting from linear flow (cm/h) to volumetric flow rates (ml/min) and vice versa ..................... 97
`
`Appendix 6
`Amino acids table ........................................................................................................................................................... 98
`
`Related literature .............................................................................................................................................. 100
`Ordering information .................................................................................................................................... 101
`
`5 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Introduction
`
`This handbook is intended for students and experienced researchers with an interest in the
`isolation of integral membrane proteins. multi protein complexes, or in refolding proteins from
`inclusion bodies. The aim is to present tools, strategies, and solutions available to meet the
`purification challenges associated with these three classes of proteins.
`
`For a background on techniques for protein purification, in general and handling recombinant
`proteins. we recommend the Recombinant Protein Purification Handbook and other
`handbooks in this series (see "Related literature" on page 100).
`
`Challenging proteins
`Our knowledge and understanding of the structural and functional biology of soluble proteins
`has increased dramatically over the last decade. Much of the technology for the production,
`purification, and analysis of soluble proteins is now at a stage where generic purification
`protocols allow relatively high success rates.
`
`The situation is different for the areas thait this handbook covers; integral membrane proteins,
`multiprotein complexes, and inclusion bodies. The need to handle and study these more
`difficult groups of proteins is clear, given that:
`
`• about 30% of a typical cell's proteins are membrane proteins, and more than 50% of the
`current drugs on the market exert their actions via membrane proteins
`
`• while carrying out their enzymatic, structural, transporting, or regulatory functions,
`proteins most often interact with each other. forming multiprotein complexes
`
`• a large proportion of normally soluble proteins that are overexpressed in E.coli end up as
`incorrectly folded and insoluble protein in inclusion bodies
`
`Outline
`After a general introduction to each area. high-level consensus workflows are presented to
`summarize current best practices in each area. Rather than providing a number of detailed
`protocols that have been optimized for individual proteins. this handbook provides general
`advice or generic protocols in a step-by-step format. The generic protocols are intended
`as starting points for development of separation protocols. Details w ill typically have to be
`changed to tailor the protocols for individual proteins. Furthermore. the required variations
`to the generic protocols cannot be predicted and unless appropriate changes are made. the
`protocols will only work poorly. if at all-this is one of the major cha.llenges for the researcher
`involved with these groups of proteins. Tc address this issue, the generic protocols are
`presented with critical parameters identified. together with ranges of values to test for the
`parameters. The handbook also provides guidance. hints. and tips when using protocols other
`than those described here.
`
`Handbook 28-9095-31 AA S
`
`6 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Comm-on acronyms and abbreviations
`
`LTAB
`
`MBP
`MPa
`M,
`MS
`Nim
`
`PBS
`pl
`
`PMSF
`psi
`PVOF
`r
`RNose
`RPC
`
`sos
`SOS-PAGE
`
`TAP
`TCEP
`
`TEV
`u
`Y2H
`
`lauryl trimethylommonium
`bromide
`maltose binding protein
`mega Pascal
`relative molecu lar weight
`mass spectrometry
`column efficiency expressed
`as theoretical plates per
`meter
`phosphate buffered saline
`isoelectric point, the pH
`at which a protein has zero
`net surface charge
`phenylmethylsulfonyl fluoride
`pounds per square inch
`polyvinylidene fluoride
`recombinant, as in rGST
`ribonuclease
`reversed phase
`chromatography
`sodium dodecyl sulfate
`sodium dodecyl sulfate
`polyacrylamide gel
`electrophoresis
`Tandem affinity purification
`Tris (2-carboxyethyl)
`phosphine hydrochloride
`Tobacco etch virus
`units (e.g., of an enzyme)
`Yeast-two-hybrid
`
`A2so
`
`CMC
`CV
`DAB
`DOM
`ONase
`OS
`
`OTT
`E.coli
`ELISA
`
`FF
`FW
`GF
`
`absorbance at specified
`wavelength (in this
`example. 280 nanometers)
`affinity chromatography
`AC
`bicinchoninic acid
`BCA
`calmodulin binding peptide
`CBP
`1-chloro-2-4-dinitrobenzene
`CDNB
`C. e/egans Caenorhabditis elegans
`CF
`chromatofocusing
`CHAPS
`3-((3-chalamidopropyl)
`dimethylammonio)-
`1-propanesulfonate
`critical micellar concentration
`column volume
`3,3'-diaminobenzidine
`dodecyl maltoside
`deoxyribonuclease
`desalting lsometimes referred
`to as buffer exchange)
`dithiothreitol
`Escherichia coli
`enzyme-linked
`immunosorbent assay
`Fast Flow
`formula weight
`gel fi ltration (sometimes
`referred to as SEC: size
`exclusion chromatography)
`green fluorescent protein
`G-protein coupled receptor
`reduced glutothione
`oxidized glutathione
`g lutathione-S-transf erase
`guonidine-HCI
`hydrophobic interaction
`chromatography
`high molecular weight
`High Performance
`horseradish peroxidase
`ion exchange
`chromatography (also seen as
`IEC in the literature)
`immobilized metal ion affinity
`chromatography
`isopropyl ~-D-thiogalactoside
`lauryldimethylamine oxide
`low molecular weight
`
`GFP
`GPCR
`GSH
`GSSG
`GST
`Guo-HCI
`HIC
`
`HMW
`HP
`HRP
`IEX
`
`IMAC
`
`IPTG
`LOAO
`LMW
`
`6 Handbook 28-9095-31 AA
`
`7 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Symbols
`
`This symbol indicates general advice to improve procedures or recommend action
`under specific situations.
`
`t
`
`This symbol denotes mandatory advice and gives a warning when special care should
`be taken.
`
`This symbol highlights troubleshooting advice to help analyze and resolve difficulties.
`
`Yellow highlights indicate chemicals, buffers, and equipment
`
`Blue highlights indicate an experimental protocol
`
`Handbook 28-9095-31 AA 7
`
`8 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Chapter 1
`Membrane proteins
`
`Introduction
`Membrane proteins play key roles in fundamental biological processes. such as transport of
`molecules, signaling, energy utilization, and maintenance of cell and tissue structures. About
`30% of the genes determined by1 genome sequencing encode membrane proteins, and these
`proteins comprise more than 50% of the current drug targets. Despite their importance, our
`knowledge of the structure and function of membrnne proteins at the molecular level lags far
`behind that for soluble proteins. For instance, at the time of the publication of this handbook,
`membrane proteins only represent around 1 % of the 3-D atomic resolution structures that
`hove been deposited in the Protein Doto Bank (http://www.pdb.org/l.
`
`Integral membrane proteins exist in o lipid environment of biological membranes
`(biomembrone). but the available techniques for purifying, handling, and analyzing them
`were optimized for water-soluble proteins in an aqueous environment. To be able to handle
`and study membrane proteins they must be dispersed in an aqueous solution. This is usuollld
`accomplished by adding a detergent that solubilizes the biomembrane and forms a soluble
`complex with the lipids and membrane proteins !Fig 1.ll. Solubilization is o harsh treatment
`that has to be carefullld optimized to avoid protein loss and inactivation. Protein denaturotion
`and/or aggregation are frequentlld encountered. Solubilization is one of the most critical
`aspects in handling membrane proteins.
`
`Other difficulties contribute to our lack of detailed structural and functional understanding of
`membrane proteins. These include:
`
`• Low abundance: The quantity of membrane proteins is often verl:J low in their natural
`setting. This makes their natural source impractical as a starting material for their
`preparation.
`
`• Difficult production: Heterologous overexpression often results in low expression levels
`and inactive protein due to insufficient membrane insertion and folding or lack of post(cid:173)
`translational modifications. Over-expression of membrane proteins can be toxic to the cell.
`
`Fig 1.1. Schematic drawing of detergent solubilizotion of membrone proteins. Membrone proteins ore transferred from
`the natural lipid bilayer (blue and yellow) to complexes with detergent (green) and, in some coses, lipids. A lipid-detergent
`micelle, a detergent micelle. and free dete,gent ore also shown.
`
`Handbook 28-9095-31 AA 9
`
`9 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Membrane protein expression, purification, and analysis present considerable challenges.
`Nevertheless, a substantial number of membrane proteins, especially from bacterial origin,
`have been over-produced, isolated, and cha.racterized in molecular detail. Also, several
`studies aiming at mopping the membrane proteome in different organisms hove been
`published. Due to great efforts in a number of membrane protein labs, generic protocols for
`membrane protein work have begun to emerge. These protocols are extremely useful as a
`starting point in the lob. In the main part of this chapter, such protocols are provided together
`with optimization advice and references for further reading.
`
`Classification of membrane proteins
`Membrane proteins are classified as peripheral or integral. Peripheral membrane proteins are
`loosely associated with the membrane and are usually water soluble after being released
`from the membrane. Peripheral membrane proteins generally present limited methodological
`challenges when compared w ith integral membrane proteins. Throughout this handbook, the
`term "membrane protein" refers to integral membrane protein unless otherwise indicated.
`Integral membrane proteins are insoluble in water. They have one or more tronsmembrone
`segments comprising polypeptide stretches that span the membrane. The transmembrone
`moiety can be constituted of a single or a bundle of a -helices or of 13-barrel-like structures
`composed of multiple polypeptide stretches. These proteins are called a -helical membrane
`proteins (Fig 1.2, left) and 13-barrel membrane proteins (Fig 1.2, right). respectively. The
`~-barrel membrane proteins are predominant in the outer membrane of Grom-negative
`bacteria and mitochondria. Some proteins display both structures.
`
`Fig 1.2. Three dimensional structure representations of on a-helical membrane protein (left: Anoboena sensory
`rhodopsin; PDB ID: lXIO: (11) and a f3-borrel membrane protein (right: ferric hydroxomote uptake receptor (fhuol from
`E. coli; PDB ID: lFCP; (Zll. The structures ore oriented such that the e~ternolly exposed area of each protein is at the
`top. The yellow lines show the approximate locations of the lipid bilayer membrane. The yellow, horizontal lines ore for
`illustration purposes only and ore not based on crystallographic data. Structures from The Protein Doto Bonk
`(http://www.pdb.org/i.
`
`10 Handbook 28-9095-31 AA
`
`10 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Purification of integral membrane proteins for structural and
`functional studies
`The high level workflow for the production and purification of integral membrane proteins
`for structural and functional studies is shown in Figure 1.3. Each of the different stages in the
`workflow is described in detail. with relevant protocols. The protocols are intended as starting
`points in the lab. Hints, tips, useful variations, and troubleshooting advice are also given. The
`focus is on protocols for bacterial membrane production and purification since this is most
`common. Protocols for eukaryotic membrane proteins are less well developed. However,
`much of the general advice is also valid for work w ith eukaryotic membrane proteins.
`
`Natural source
`
`Cloning and expression
`
`~
`
`!
`
`0
`
`Expression screening
`
`0 I Detergent screening
`
`Cell harvest
`
`!
`
`Ce I disruption and
`membrane prep
`
`!
`
`Solubillzat1on
`
`!
`
`Purification
`
`!
`
`Purity ond
`homogeneity check
`
`!
`
`Conditioning
`
`!
`
`Structural and/or
`functional studies
`
`Fig 1.3. Workflow overview for membrane protein isolation ond purificotion for structural and functionol studies.
`
`Handbook 28-9095-31 AA 11
`
`11 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Starting material
`Membrane proteins from natural sources
`The natural source of a membrane protein can be considered as a starting material for
`purification. The only three-dimensional structure in molecular detail that has been reported
`to date for a eukaryotic G-protein coupled receptor (GPCR). bovine rhodopsin, was obtained
`with prot,ein purified from bovine retina, where the protein is highly abundant (8). In many
`coses, however; low abundance of the target protein precludes the use of the natural source
`as starting material.
`
`Examples of purifications from natural sources ore presented later in this chapter.
`
`Cloning
`Vectors used for the expression of soluble proteins ore also commonly used for the
`production of membrane proteins. It is useful to design a number (10 to 50) of different
`constructs, including different homologues, to increase the chance that a particular
`membrane protein can be produced in an active form.
`
`In addition to the general considerations for choosing a vector (see Recombinant Protein
`Purification Handbook, in "Related literature" on page 100). a number of other aspects relate
`more specifically to choosing a vector for expressing membrane proteins.
`
`-
`
`Affinity togging greatly facilitates expression screening based on chromatographic
`enrichment, as well as optimization and use of protocols for purification of membrane
`proteins. Polyhistidine tags are commonly used for membrane proteins, but the GST(cid:173)
`tag and others have also been used successfully. The insertion of a protease cleavage
`site between the affinity tag and the target protein enables removal of the tag before
`further analyses.
`
`While a hexahistidine tag (His6) is the standard option for water-soluble proteins,
`longer histidine tags (with 8 or 10 histidine residues) are often used for membrane
`proteins to increase the binding strength and thus improve yields in IMAC purification.
`Drawbacks with longer (> 6 histidine residues) histidine tags are that expression
`levels have been reported to be decreased in some cases and that a higher imidazole
`concentration is required for elution.
`
`Tags should generally be placed on t he ( -terminal end of the protein to reduce risk of
`affecting the membrane insertion process based on the N-terminal signal peptide.
`
`Fusion of the target membrane protein to a fluorescent protein tag such as GFP
`in combination with a histidine tog allows direct and convenient visualization of
`the target during expression, solubilization, and purification and can speed up the
`optimization of these processes (6).
`
`12 Handbook 28-9095-31 AA
`
`12 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Expression and screening
`To correctly decide which conditions and constructs will be best suited for producing the
`protein for the intended study, an efficient screening protocol is essential. Because of the
`relatively low concentrations of overexpressed membrane proteins, it is useful to apply
`affinity togs com bined with separation methods that allow enrichm ent of the target protein.
`
`Overexpression is a major bottleneck in the overa ll workflow for membrane protein
`production. Several host systems are avaiilable and the final choice will depend both on
`protein-specific requirements (e.g., for post-translational modifications) and practical aspects
`(e.g., available equipment in the lab and expertise). It is often useful to try different hosts or
`host strains in parallel for a particular target protein to increase the likelihood of success. In
`addition, homologous membrane proteins from several sources can be cloned in parallel to
`be able to select those that express well.
`
`E.coli strain BL21 (DE3) is the most comrronly used host for overexpression of membrane
`proteins, in combination with a pET vector. "High" expression levels for functional membrane
`proteins are usually more than an order of magnitude lower than for overexpression of
`water-soluble proteins in E. coli. One inherent issue is that membrane proteins need to be
`inserted into membranes, and the availability of membrane structu res in most cells is limited.
`
`The issue with limited membrane availability can be addressed by using a host w ith large
`amounts of internal membranes (e.g., Rhodobacter spp.; (3)). Anothe r way of avoiding the
`limitations set by available membranes is to produce the membrane protein as inclusion
`bodies. This is usually not desired but may allow preparation of active protein through
`salubilization of the inclusion bodies using denaturants followed by refolding. Successful
`refolding of ~-barrel membrane proteins from inclusion bodies has been achieved (4) but
`refolding of a-helical membrane proteins is an even greater challenge. For a separate
`discussion on inclusion bodies. see Chapter 3.
`
`A modest growth and expression rate is beneficial to avoid the formation of inclusion
`bodies when using E. coli as a host. This can be achieved by the use of a weak
`promoter, a low concentration of inducer and/or lowering the growth temperature
`after induction.
`
`An overview of different expression systems for membrane proteins is provided in Tobie 1.1.
`For a review on important considerations for membrane protein expression, see reference 7.
`
`Hondbook 28-9095-31 AA 13
`
`13 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Tobie 1.1. Overexpression systems used for prokaryotic ond eukar\jotic membrane protein production
`
`Expression system
`
`Advantages
`
`Disadvantages
`
`E. coli
`
`Yeast
`
`The most widely used
`overexpression system for
`(prokaryotic) membrane protein
`production.
`
`Often not suitable for overexpression of
`eukoryotic membrane proteins
`
`No glycosylation and limited post(cid:173)
`translational modifications
`
`Can perform some post(cid:173)
`translational modifications
`
`Does not produce high cell densities
`IS. cerevisiae)
`
`Severo\ different yeast systems
`have been used for membrane
`protein production (SI
`
`Hyperglycosylotion con occur
`15. cerevisioe)
`
`Different lipids lcompored with mammalian
`cells)
`
`Insect cells
`
`Less complex growth
`conditions compared with
`mammalian cells
`
`More costly and complex than E.coli or
`yeast; different lipids (compared with
`mammalian cells)
`
`Mammalian cells
`
`Rhodobacter spp.
`
`Cell free
`
`Relatively high expression
`levels
`
`Glycosylotion
`
`CHO, BHK and other cell lines
`are often used for functional
`studies of receptors
`
`Authentic (mammalian) protein
`is produced
`
`High e.xpression levels through
`coordinated synthesis of
`foreign membrane proteins
`with synthesis of new internal
`membranes 13)
`
`Allows expression of toxic
`proteins and proteins that are
`easily degraded in vivo
`
`Allows incorporation of labeled
`and non-natural amino acids.
`
`High cost and complex work
`
`Different lipids (compared with mammalian
`cells)
`
`High cost
`
`Membrane protein insertion in membrane
`or detergent micelle hos not been fully
`developed
`
`Disposable 96-well filter plates, from GE Healthcare, prepacked with affinity purification
`media for histidine- or GST-tagged proteins can be used for reproducible, high-throughput
`screening of protein expression. Typical applications include expression screening of d ifferent
`constructs, screening for suitable detergents and solubility of proteins, and optim ization of
`t he conditions for small-scale parallel purificat ion. Plates are availa ble prepacked w ith Ni
`Sepharose™ High Performance or Ni Sepharose 6 Fast Flow for working with histidine-tagged
`proteins (His MultiTrap™ HP or His MultiTrap FF, respectively); and Glutathione Sepharose 4
`Fast Flow o r Glutathione Se pharose 4B for working with GST-tagged proteins {GST MultiTrop
`FF or GST MultiTrap 4B, respectively).
`
`14 Handbook 28-9095-31 AA
`
`14 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Small-scale expression screening of histidine-tagged membrane
`proteins from E. coli lysates
`
`Cell lysis and solubilization
`
`Buffer preparation
`
`Lysis buffer: 20 mM sodium phosphate, 100 mM NaCl. 2 mM MgCI,. 20 mM imidazole. 0.5 mM Tris
`I2-corboxyethyl) phosphine hydrochloride (TCEP). 5 u/ml benzonase.
`1 mg/ml lysozyme. EDTA-free protease inhibitor cocktail, (concentration according
`to manufacturer's recommendation). 1-2% ofa selection of detergents. pH 7.4
`
`Procedure
`
`1. Harvest cells from the culture by centrifugation at 7000 to 8000 x g for 10 min or at 1000 to
`1500 x g for 30 min at 4°C.
`
`2. Discord the supernatant. Place the bacterial pellet on ice.
`
`3. Suspend the bacterial pellet by adding 5 to 10 ml of lysis buffer for each gram of wet cells.
`To prevent the binding of host cell proteins with exposed histidines, it is essential to include
`imidazole at a low concent ration in the sample and binding buffer.
`
`4. Leave for 2 h with mild agitation at room temperature or 4°C, depending on the sensitivity of
`the target protein.
`
`5. Measure and adjust pH if needed.
`
`Expression screening procedure
`
`Materials
`
`His MultiTrop HP or His MultiTrap FF
`
`Centrifuge that handles 96-well plates
`
`Buffer preparation
`
`Binding buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM imidozole, 0.5 mM TCEP,
`1 to 2% detergent, pH 7.4. IThe optimal imidozole concentration is protein
`dependent; 20 to 40 mM is suitable for many proteins.)
`
`Wash buffer: 20 mM sodium phosphate, 500 mM NaCl, 20 to 40 mM imidazole, 0.5 mM TCEP,
`0.03% dodecyl moltoside IDDM), 1 to 2% detergent, pH 7 .4,
`
`Elution buffer: 20 mM sodium phosphate. 500 mM NaCl. 500 mM imidozole, 0.5 mM TCEP, 0.03%
`DOM, 1 to 2% detergent, pH 7.4
`
`To increase the purity, use as high a concentration of imidazole as possible in the
`sample and binding buffers without losing binding capacity.
`
`Handbook 28-9095-31 AA 15
`
`15 of 107
`
`Fresenius Kabi
`Exhibit 1037
`
`

`

`Preparing the filter plate
`
`1. Peel off the bottom seal from the 96-well filter plate. Be sure to hold the filter plate over o sink
`to accommodate ony leokoge of storage solution when removing the bottom seal.
`
`2. Hold the filter plate upside down ond gently shake it to dislodge ony medium adhering to the
`top seal. Return the filter plate to an upright position.
`
`3. Place the filter plate against the bench surface and peel off the top seal.
`
`4. Position the filter plate on top of o collection plate.
`
`Note: Remember to change or empty the collection plate as necessary during the following
`steps.
`5. Centrifuge the filter plate for 2 min at 200 x g to remove the ethanol storage solution from the
`medium.
`
`6. Add 500 µI of deionized water to each well. Centrifu~e the plate for 2 min at 2