Service package 1: Protein production
The Norwegian Structural biology center (NorStruct) offers three service packages:
SP1: Protein production
SP2: Structure determination
SP3: Drug discovery and design
These are outlined in the boxes to the right.
In SP1 (Protein production) NorStruct offers unique
opportunities for subcloning, small and pilot scale
expressions (up to 10 L) and purification, in addition to
protein expression optimization and refolding.
The subcloning, small scale expression and purification
using E. coli as expression host will be performed in high
throughput format. Expression will also be offered using
Pichia pastoris as expression host. The customer can
select between N- and C-terminal His-tag. Expression of
target proteins will be verified by SDS-PAGE and MS. More details of the standard
commissioning in SP1 can be obtained from the NorStruct web site http://uit.no/norstruct/ or by
contacting NorStruct.
Table 1 Standard commissioning in SP2 Structure determination.a
Service
Package (SP)
SP1.1a
SP1.1b
SP1.1c
SP1.1d
SP1.2a
Task
In E. coli:
Cloning, small scale
expression and
purification.b
In E. coli:
Medium scale-up
expression and
purification.b
In E. coli:
Large scale-up expression
and purification.b
In E. coli
Protein expression
optimization b
Specification
Intracellular proteins: Cloning of one construct with N- or C-terminal His-tag.
Extracellular proteins: Cloning of one construct with C-terminal His-tag using the
pET-26b(+) vector.
Transformation of construct into 3 different E. coli strains.a Expression of 2 ml and
10 ml cultures for intracellular and periplasmic proteins, respectively, at 20°C.
Purification using Ni-NTA columns.
One litre expression of the most promising strain from SP1.1a. Alternatively,
expression of a desired gene in expression strain supplied by the customer.
Expression at 20C. Purification using HisTrap columns.
Ten litre expression of the most promising strain from SP1.1a. Alternatively,
expression of a desired gene in expression strain supplied by the customer.
Expression at 20C. Purification using HisTrap columns.
Cloning: Construct cloned into 8 vectors and transformed into E. coli DH5.
Screening of 3 colonies from each. One construct (from each of the 8) transformed
into 3 strains.
Expression: One transformant of each for 2 ml expression at 20C and 37C.
Media optimization: Choose the three strains with best expression and test 4
media.
One construct with N- or C-terminal His-tag. Expression in 0.5 L. Purification using
HisTrap columns.
In P. pastoris:
Cloning, screening, small
scale expression and
purification.
Ten litre expression of the most promising strain from SP1.2a. Purification using
SP1.2b
In P. pastoris:
Large scale-up expression HisTrap columns.
and purification.
SP1.3
Protein refolding: Screen
Refolding using commercially available 96-kit (purified inclusion bodies must be
to find refolding condition
supplied by customer).
for insoluble proteins.
aSee SP2 Protein production web pages for terms for delivery: http://uit.no/norstruct/3195/6
bSee footnotes on SP2 Protein production web pages for specifications: http://uit.no/norstruct/3195/6
The Norwegian Structural Biology Centre
University of Tromsø, N-9037 Tromsø, Telephone: (+47) 77 64 40 00, Fax (+47) 77 64 47 65
Direct telephone: (+47) 77 64 40 70, E-mail: norstruct@chem.uit.no
http://uit.no/norstruct/
Preliminary order form Service package 1:
Protein production.

Please place tick marks to indicate which tasks are desired. For simplicity this icon
can be
copied and replaced by the empty boxes. Then return this electronic form by E-mail to NorStruct,
and you will receive in response a tailored order form and prizes for placing the final order.
NorStruct will not be able to handle genes originating from organisms belonging to risk class 3 or
4.
Order:
Date:
…../…../20……
Title (and NorStruct project number if
available):
Customer:
Institution:
Adress:
Contact person:
E-mail:
Phone:
Order:
(Tick)
SP
Task
SP1.1a
In E. coli:
Cloning, small scale expression and
purification
In E. coli:
Medium scale-up expression and
purification
In E. coli:
Large scale-up expression and
purification.
In E. coli
Protein expression optimization
In P. pastoris:
Cloning, screening, small scale expression
and purification.
In P. pastoris:
Large scale-up expression and
purification.
Protein refolding: Screen to find refolding
condition for insoluble proteins.
SP1.1b
SP1.1c
SP1.1d
SP1.2a
SP1.2b
SP1.3
.
The Norwegian Structural Biology Centre
University of Tromsø, N-9037 Tromsø, Telephone (+47) 77 64 40 00, Fax (+47) 77 64 47 65
Direct telephone (+47) 77 64 40 70, E-mail: norstruct@chem.uit.no
http://uit.no/norstruct/
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General information
Details
Information
Protein name:
Function of protein:
protease, DNA binding, etc.
………………..
………………..………………..………………..
………………..………………..………………..
DNA details:
Number of nucleotides:
Sequence:
………………..
Please paste the nucleotide sequence here:
Protein details:
Number of amino acids:
Molecular weight (kDa):
GenBank accession number:
pI (theoretical or measured):
Sequence:
………………..
………………..
………………..
………………..
Please paste the amino acid sequence here:
Construct details:
Does the construct contain
tags:
If yes, how many, which type
and which order:
Does the construct contain
TEV cleavage site:
Original source organism:
Risk class for this sourcea:
Risk class of proteina:
yes
no
…………………………………………………………
…………………………………………………………
yes
no
………………..………………..
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a viral protein;
a toxin;
a prion protein;
a virulence factor;
other,
no risk.
The customer must provide NorStruct with ALL
necessary information about the risk classes of the
original source and of the produced protein to take all
necessary safety precautions, to avoid any harm to
the environment and/or people.
a
NorStruct may not be able to handle genes originating from organisms that belong to risk class 3 or 4. For proteins being
viral proteins, toxins, prion proteins or virulence factors, NorStruct might have restrictions handling such proteins. Possible
risk factors and the feasibility of handling such high risk class projects will be dealt with in close collaboration with each
customer.
For more details on the risk classes, please consult the web page (in Norwegian) about protection concerning biological
risk factors: http://www.lovdata.no/cgi-wift/ldles?doc=/sf/sf/sf-19971219-1322.html
Cloning and small scale expression and purification details
Details
Information
The Norwegian Structural Biology Centre
University of Tromsø, N-9037 Tromsø, Telephone (+47) 77 64 40 00, Fax (+47) 77 64 47 65
Direct telephone (+47) 77 64 40 70, E-mail: norstruct@chem.uit.no
http://uit.no/norstruct/
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Customer provides:
Sub-cloned construct
Delivery:
Vector/system: ………………..
Genomic DNA
cDNA (obligatory for eukaryotic proteins)
Other details:
……………………………………………………………..
Small scale expression type
(SP1.1a, SP1.1d, SP1.2a):
Intracellular protein in E. coli with:
N-Term. His-tag
C-Term. His-tag
Extracellular protein in E. coli with:
C-Term. His-tag
Expression in P. pastoris:
Protein expression optimization
(SP1.1d):
Vectors to be used or
truncated forms of the
construct (up to 8):
Strains to be used (up to 3):
Temperature to be used:
Media to be used:
8 vectors (specify in Additional information section
at the end of the form)
Truncated forms (please provide details in the
Additional information section at the end of the
form):
……………….. ……………….. ………………..
………………..
………………..
The customer’s suggestion for
primer details:
Antibodies and or assay:
Protein specific antibodies will be provided
Activity assay will be provided
Other specifications
Medium and large scale protein production details
Details
Information
Delivery:
Customer provides:
Stab culture containing transformed bacterial culture
Excised agarose gel containing transformed bacterial
culture
Other details:
……………………………………………………………..
Production type:
Medium scale
Large scale
The Norwegian Structural Biology Centre
University of Tromsø, N-9037 Tromsø, Telephone (+47) 77 64 40 00, Fax (+47) 77 64 47 65
Direct telephone (+47) 77 64 40 70, E-mail: norstruct@chem.uit.no
http://uit.no/norstruct/
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Small scale expression details:
Vector and strain:
Temperature:
Media:
Induction details:
Proof:
Small scale purification details:
Chromatographic method
(His Trap, ionic exchange,
gelfiltration etc.):
Temperature:
Buffers (type(s) and pH):
Proof:
Antibodies and or assay:
……………….. ………………..
………………..
………………..
………………..
Please insert picture of SDS gel here (obligatory):
Please insert MS results (if available) here:
………………..
………………..
………………..
Please insert picture of chromatogram(s) here
(obligatory):
Please insert picture of SDS gel of purified protein
(obligatory):
Protein specific antibodies will be provided
Activity assay will be provided
Other specifications:
Additional protein characteristics.
In order to obtain soluble protein it is advisable to perform sequence analysis that can aid us in
the protein expression and purification stages. We encourage all our customers to perform and
report such an analysis.
.
Factors
Question
Information
Glycosylation
1)
2)
Cysteines
1)
2)
Metal binding
1)
2)
3)
Is glycosylation known for this
protein?
Is this something to consider?
1) ………………..
Are there free cysteines in this
protein (if yes; how many)?
Should reducing agents be
considered during purification?
1) ………………..
Are there any metal binding sites?
If yes, which and how many?
(e.g. Fe, Mg, Mn, Zn, etc)
Are there any metal binding
motifs? Zink fingers, CxxC, [Fe-S],
etc?
1) ………………..
2) ………………..
2) ………………..
2)
Nol
If yes, which reducing
agent:
-mercaptoethanol
DTT
Other, specify:……
3) ………………..
The Norwegian Structural Biology Centre
University of Tromsø, N-9037 Tromsø, Telephone (+47) 77 64 40 00, Fax (+47) 77 64 47 65
Direct telephone (+47) 77 64 40 70, E-mail: norstruct@chem.uit.no
http://uit.no/norstruct/
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Co-factor
1)
Could there be any co-factor
binding sites; ATP, NAD(P)+, FMN,
others?
1) ………………..
If yes, should this be added
in to the buffer?
Yes
No
Membrane
1)
Is it possible that all or part of the
protein could be membrane
attached?
Could trans-membrane elements
be removed to increase the protein
solubility?
1) ………………..
2)
2) ………………..
Signal peptides
1)
2)
Are there any signal sequences?
Should this possibly be removed?
1) ………………..
2) ………………..
Domains
1)
Are there multiple domains? If yes
please specify residue range.
1) ………………..
Oligomerisation
1)
Is oligomerisation known for this
protein, dimer, trimer etc.?
1) ………………..
Additional information
Please provide us with:
1) Construct details for SP1.1d
2) Any additional information about the protein since this can be critical for the success of
the cloning, expression and protein purification.
Additional details about cloning,
expression, purification:
Information
Additional contact persons
In case of NorStruct would like to request more details please provide us with the name of contact
person(s) involved in the project that can help us with additional information.
Name
Address
E-mail
Phone
The Norwegian Structural Biology Centre
University of Tromsø, N-9037 Tromsø, Telephone (+47) 77 64 40 00, Fax (+47) 77 64 47 65
Direct telephone (+47) 77 64 40 70, E-mail: norstruct@chem.uit.no
http://uit.no/norstruct/
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