Module 2 Overview: Spring Break

1
Module 2 overview
lecture lab
1. Introduction to the module 1. Start-up protein eng.
2. Rational protein design 2. Site-directed mutagenesis
3. Fluorescence and sensors 3. DNA amplification
4. Protein expression 4. Prepare expression system
SPRING BREAK
5. Review & gene analysis 5. Gene analysis & induction

6. Purification and protein analysis 6. Characterize expression
7. Binding & affinity measurements 7. Assay protein behavior
8. High throughput engineering 8. Data analysis
2
Lecture 6: Protein purification
I. Standard purification methods

A. Harvesting and lysis
B. Protein separation techniques
II. Assessing purified proteins

A. Electrophoresis
B. Mass spectrometry
C. Protein sequencing and AA analysis
Courtesy of David S Goodsell. Used with permission.
3
4
Once weʼve collected the
cells, how do we get the
proteins out?
Photos removed due to copyright restrictions.

Three laboratory devices:
* Blender
* French press
* Sonicator
Image of cells undergoing lysis
clockwise from top left:

www.biomembranes.nl
bioinfo.bact.wisc.edu
matcmadison.edu
5

www.biomedcentral.com
Image from Rekha, N., and N. Srinivasan. BMC Structural Biology 2 (2003): 4.
http://www.biomedcentral.com/1472-6807/3/4
Courtesy of the authors, © 2003 Rekha and Srinivasan.
6
Separation techniques
most common, e.g. Ni-NTA
in addition to
affinity
Source: GE Healthcare Gel Filtration Principles and Methods handbook.

http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/LD_153206006-R350?OpenDocument&hometitle=search
© GE Healthcare. All rights reserved. This content is excluded from our Creative Commons license.
For more information, see http://ocw.mit.edu/fairuse
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8
Nickel affinity purification with Ni-NTA agarose
9
Many other tags can be used for protein purification:
tag residues matrix elution condition
poly-His ~6 Ni-NTA imidazole, low pH
FLAG 8 anti-FLAG antibody low pH, 2-5 mM EDTA
c-myc 11 anti-myc antibody low pH
strep-tag 8 modified streptavidin 2.5 mM desthiobiotin
CBP 26 calmodulin EGTA, EDTA
GST 211 glutathione reduced glutathione
MBP 396 amylose 10 mM maltose
Tags may be chosen because they

• interfere minimally with protein structure/function
• improve recombinant protein expression or solubility
• offer most convenient purification methods
All tags may be cleaved from expressed proteins using specific

proteases, if desired.
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Gel filtration (size exclusion chromatography) principle
Source: GE Healthcare Gel Filtration Principles and Methods handbook.

http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/LD_153206006-R350?OpenDocument&hometitle=search
© GE Healthcare. All rights reserved. This content is excluded from our Creative Commons license.
11
Quantification of purified proteins
use Beer-Lambert law:

A280 = ε280cl
ε280 is the extinction

1.2 coefficient; it can be
I
determined rigorously,
Absorbance
0.9
or estimated:
0.6 B ε280 ~ nW x 5500
+ nY x 1490
0.3
+ nC x 125
G
240 260 280 300 320
Wavelength (nm)
Image by MIT OpenCourseWare.

12
Assessing proteins for identity and purity
Most standard technique is sodium dodecylsulfate polyacrylamide gel

electrophoresis (SDS-PAGE):
• basis is the tendency of proteins to unfold in SDS and bind a
fixed amount SDS per protein (1.4 g/g)
• negative charge of SDS overwhelms protein charges

• proteins have same charge to mass ratio, but are differentially
retarded by the separation gel
• stacking layer “focuses” proteins before separation layer
glycine ions
pH 6.8
pH 8.8 chloride ions
Source: "Multiphasic Buffer Systems" (http://nationaldiagnostics.com/article_info.php/articles_id/10).

© National Diagnostics U.S.A.. All rights reserved. This content is excluded from our Creative Commons license.
13
Coomassie brilliant blue staining
• binds proteins primarily via aromatic

residues and arginine
• undergoes absorbance shift from 465
nm (brownish) to 595 nm (blue)
• basis for Bradford Assay; can be used
to quantify proteins over ~3 kD
http://www.eiroforum.org/media/gallery/embl.php
Courtesy of EMBL. Used with permission.
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SDS-PAGE gives an approximate MW and purity estimate, but how can

we be sure the protein weʼve purified is the correct one?
• activity assay if one is available
• knowledge of exact mass (mass spectrometry)
• N-term. sequencing and AA analysis, if necessary
Image: public domain (USGS)

Source: http://www.kcl.ac.uk/research/facilities/mspec/instr/maldi-tof-introa.html
© Kings College London / Centre of Excellence for Mass Spectrometry.
All rights reserved. This content is excluded from our Creative Commons license.
15
N-terminal sequencing (Edman degradation)
• products identified by chroma-

tography or electrophoresis
• typically ~5 cycles practical for
routine N-term. sequencing
en.wikipedia.org/wiki/Edman_degradation
public domain image
Amino acid analysis
• HCl digestion to digest

peptide bonds
• HPLC to quantify AA
components
MIT OpenCourseWare
http://ocw.mit.edu
20.109 Laboratory Fundamentals in Biological Engineering

Spring 2010
For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms.

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1

5. Review & gene analysis 5. Gene analysis & induction

Lecture 6: Protein puriﬁcation

I. Standard puriﬁcation methods

II. Assessing puriﬁed proteins

cells, how do we get the

Photos removed due to copyright restrictions.

clockwise from top left:

most common, e.g. Ni-NTA

Source: GE Healthcare Gel Filtration Principles and Methods handbook.

tag residues matrix elution condition

poly-His ~6 Ni-NTA imidazole, low pH

FLAG 8 anti-FLAG antibody low pH, 2-5 mM EDTA

c-myc 11 anti-myc antibody low pH

strep-tag 8 modiﬁed streptavidin 2.5 mM desthiobiotin

CBP 26 calmodulin EGTA, EDTA

GST 211 glutathione reduced glutathione

MBP 396 amylose 10 mM maltose

Tags may be chosen because they

• offer most convenient puriﬁcation methods

All tags may be cleaved from expressed proteins using speciﬁc

Source: GE Healthcare Gel Filtration Principles and Methods handbook.

use Beer-Lambert law:

ε280 is the extinction

Image by MIT OpenCourseWare.

Most standard technique is sodium dodecylsulfate polyacrylamide gel

ﬁxed amount SDS per protein (1.4 g/g)

• negative charge of SDS overwhelms protein charges

retarded by the separation gel

• stacking layer “focuses” proteins before separation layer

pH 8.8 chloride ions

Source: "Multiphasic Buffer Systems" (http://nationaldiagnostics.com/article_info.php/articles_id/10).

• binds proteins primarily via aromatic

SDS-PAGE gives an approximate MW and purity estimate, but how can

Image: public domain (USGS)

• products identiﬁed by chroma-

Amino acid analysis

• HCl digestion to digest

20.109 Laboratory Fundamentals in Biological Engineering

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