Separation of Proteins by gel electrophoresis · 2012-03-11 · All t DNA ith l d tAllow vector DNA with cloned gene to multiply Select clones by selectable marker(s) (antibiotic

Separation of Proteins by gel electrophoresis

Polyacrylamide (polymer of acrylamide) gel is a chemical (not biological) matrixPolyacrylamide (polymer of acrylamide) gel is a chemical (not biological) matrixUsed for separation of biomolecules such as proteins or DNA fragmentsAbility to resolve DNA fragments differing by a single base It is also used for separation of proteins based on their molecular weights (1D SDS PAGE)PAGE)OR first by their charge (pI) using isoelectric focusing (IEF) (also known as first dimension) and then by molecular weight (Mr) (also known as second dimension SDSPAGE). This method of separation of proteins is called O’Farrell 2-D PAGE, whichgenerates a comprehensive “protein fingerprint” or “proteome analysis” of expressedgenerates a comprehensive protein fingerprint or proteome analysis of expressedproteins in a living culture. The 2-D protein gel method has the unique capacity for theresolution of complex mixtures of proteins, permitting the simultaneous analysis ofhundreds or even thousands of gene productsThe separated proteins can be transferred on a nitrocellulose or PVDF membrane andThe separated proteins can be transferred on a nitrocellulose or PVDF membrane andprobed with antibodies for identification of the expression of a specific protein. Thismethod of identification of a protein is called the western blotThe immobilized protein on the PVDF membrane can be subjected to protein

mcirosequencingmcirosequencing

NOTE: Unpolymerized acrylamide (polyacrylamide) is a neurotoxin; therefore safe laboratory practice should be adopted when used for PAGE gel electrophoresis.

(References for 2-D PAGE: (1) O' Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007-4021. (2) O'Farrell PZ, Goodman HM, O'Farrell PH (1977) High resolution two-dimensional electro-phoresis of basic as well as acidic proteins. Cell 12: 1133-1142).

PROTEIN MICROSEQUENCING

The two major direct methods of protein sequencing areThe two major direct methods of protein sequencing are

(1) mass spectrometry(2) the Edman degradation reaction(3)It i l ibl t t i id f th DNA(3)It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein

Edman degradation: it allows the ordered amino acid composition of a g pprotein. It can resolve peptides up to approximately 50 amino acids long.

First, long protein chains are broken up into small fragments which can then be sequenced individually Digestion is done either by:be sequenced individually. Digestion is done either by:

(A)endopeptidases such as trypsin or pepsin(B) by chemical reagents such as cyanogen bromide

Limitations of the Edman degradation method:

Because the Edman degradation proceeds from the N-terminus of the proteinBecause the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminal amino acid has been chemically modified or if it is concealed within the body of the protein.

35S-methionine Labeling of Serum-induced ProteinsC k i 9 i i l di (O 0 2)1 2 C. jeikeium in M9 minimal medium (OD450 = 0.2)

↓ ↓M9 medium M9 medium + serum

0

0.2

0.4

0.6

0.8

1

1.2

0 hr

1 hr

2 hr

3 hr

4 hr

5 hr

6 hr

7 hr

8 hr

16hr

Ti

OD

450 M9

M9+10% serum

↓1µl of 35S-met (10 µCi/ml.) was added to 1 ml. of each of the above cultures

↓

Time

↓

Incubate at 170 rpm, 37 °C

↓↓Centrifuge cells, wash 3x in PBS (pH 7.2)

↓R d ll i 20 l PBS ( H 7 2)Resuspend cells in 20 µl. PBS (pH 7.2)

↓Cells were lysed by addition of SDS gel loading dye and heating to 100°C for 20 min.y y g g y g

4 hrs overnight

36 kD

No 1% 10% No 1% 1% 10%serum serum serum serum serum

CSIP 36: AlaProAlaGlyCysLeuGlyGlyLeuCSIP-36: AlaProAlaGlyCysLeuGlyGlyLeu

NH-terminal: Ala Pro Ala Gly Cys Leu Gly Gly Leu (Determined by Edman Degradation method)A P A G C L G G L

gcg ccg gcg ggy wgc ttg ggy ggy ttg( t t ) (D t P b )(y = c or t; w= t or a; ) (Degenerate Probe)

Single-Letter Amino Acid Code

G - Glycine (Gly)Mixed base definitions

G Glycine (Gly) •P - Proline (Pro) •A - Alanine (Ala) •V - Valine (Val) L L i (L )

R A, G

Y C, T

•L - Leucine (Leu) •I - Isoleucine (Ile) •M - Methionine (Met) •C - Cysteine (Cys)

M A, C

K G, T y ( y )

•F - Phenylalanine (Phe) •Y - Tyrosine (Tyr) •W - Tryptophan (Trp) •H Histidine (His)

S C, G

W A, T

H A C T •H - Histidine (His) •K - Lysine (Lys) •R - Arginine (Arg) •Q - Glutamine (Gln)

H A, C, T

B C, G, T

V A C G •N - Asparagine (Asn) •E - Glutamic Acid (Glu) •D - Aspartic Acid (Asp) •S Serine (Ser)

V A, C, G

D A, G, T

N A, C, G, T •S - Serine (Ser)•T - Threonine (Thr)

, , ,

Study Gene Structure, Function and Biotechnology Applications using Recombinant DNA Technology

Identification of a biological function regulated by a protein and its geneIdentification of a biological function regulated by a protein and its gene

Be able to identify differentially expressed protein following treatment or involved in a physiological/biochemical function if the cell (Tool-Gel electrophoresis)

Be able to identify, characterize and sequence (partial) the protein (Tool – Protein microsequencing)

Be able to convert the protein (partial) sequence into DNA sequence (degenerate sequence) (Tool- (a) Use of DNA software; (b) codon table; and (c) codon usage table)

Be able to synthesize the deduced DNA sequence to be sued a probe forBe able to synthesize the deduced DNA sequence to be sued a probe for hybridization (Tool- (a) DNA purification; (b) Nucleic acid hybridization)

OR PCR method to “pull-out” the gene sequence from the genome (Tool-PCR)

Be able to clone the gene fragment on a plasmid (Tool- (a) Vectors; (b) DNA-RNA dif i )modifying enzymes)

Be able to transfer the cloned gene and screen for the clone (Tool-Colony/plaque hybridization OR other biochemical methods

Be able to express and isolate the recombinant protein (Tool-plasmid expression p p ( p pvector)

Be able to generate “knock-out” mutations for the gene and study complementation to establish the designated function of the cloned gene (Tool-Site-directed mutagenesis; PCR-generated mutations; suicide vector-mediated mutagenesis; reciprocal recombination; transposon-mediated mutagenesis

Bed able to study transcriptional and translational regulation of the cloned gene (Tool-Norther blot; western blot)

Tools for Recombinant DNA Technology

(cloning of the serum-induced growth gene factor from CJK)Protein gel electrophoresis and protein microsequencing

Use of codon table and codon preference (usage) tableUse of codon table and codon preference (usage) table

DNA purification

Concentration and purity of DNA

Gel electrophoresis

Restriction endonucleases

Gene library

DNA-DNA hybridization

Subcloning of the gene

DNA sequencingDNA sequencing

Use of software for gene analysis

Gene expression study

Basic Tools for Recombinant DNA Technology

The basic steps in Gene cloning: Plasmid and the gene fragment to be cloned are treated with restrictioncloned are treated with restriction endonuclease(s)

These fragments are ligated by ligase enzyme using a co-factor (ATP for T4 bacteriophage ligase; NAD for E. colibacteriophage ligase; NAD for E. coli ligase)

Transfer clones in E. coli by Transformation

All t DNA ith l d tAllow vector DNA with cloned gene to multiply

Select clones by selectable marker(s) (antibiotic resistance gene) and phenotypic characteristics of the clonedphenotypic characteristics of the cloned gene

Gene cloning and DNA analysis-An Introduction by T.A. Brown, Blackwell Publishing – Chapter 1


PCR method –DNA amplification

-follows the principles of DNA replication

-primers

-Targeted gene

-dNTP

-salt buffer with MgCl2

-a thermal cycler


Tools for Recombinant DNA TechnologyGene isolation by cloning followed by selection and screening of the cloned gene. A selectable marker or a phenotypic characteristic is required for screening for the cloned gene. If such marker is not available then identification of the clone q g gcould be difficult and tedious. This method of gene cloning is referred as “shot-gun” approach. Alternatively, a “gene library” can be used for such method of cloning


Tools for Recombinant DNA TechnologyTechnology

Gene isolation by PCR

At least a part of the gene sequence must be available. That could be obtained either from the partial protein sequence OR a similar gene from another organism.

A selectable marker or a phenotypic characteristic is required for screening the cloned gene. If such marker characteristic of the targeted gene is not available then identification of the clone could be difficult and tedious Howevercould be difficult and tedious. However, since the primers used are targeted gene specific, it is likely that the targeted gene is amplified by this method PCR could be useful to clone a

l ti lgene relatively easy

PCR can be used to clone a gene from a small amount of targeted DNA

Millions of copies of the amplified p pgene can be obtained by PCR



Plasmids

(a) Natural plasmids (large -~70-100 kbp)p)

(b) Recombinant (chimeric) plasmids (small - <1 kbp-~10 kbp)p)

(c) Selectable markers on plasmids


Tools for Recombinant DNA TechnologyPlasmids

(a) Conjugation and compatibility/non-compatibility of plasmids

(b) Integrative plasmids

(c) Non-integrative plasmids


Tools for Recombinant DNA TechnologyPlasmids

( ) Si d b f l id(a) Size and copy number of plasmids

(b) Plasmid classification




Bacteriophages (for recombinant DNA technology)

(a) Bacteriophage structures

(b) Lytic phages (bacteriophage Lambda head-and-tail – used for gene library)g y)

(c) Lysogenic phages (bacteriophage Lambda – used for gene library)

(d) Non-lytic phage (bacteriophage M13 filamentous – used for gene sequencing)

The infection cycles of bacteriophage LambdaGene cloning and DNA analysis-An Introduction by

T.A. Brown, Blackwell Publishing – Chapter 2

Tools for Recombinant DNA TechnologyNon-lytic phage (Filamentous bacteriophage M13 – used for gene cloning and sequencing)

The infection cycles of bacteriophage M13


Tools for Recombinant DNA TechnologyBacteriophages (for recombinant DNA technology): Bacteriophage Lambda – used for gene library)

The Lambda genetic map showing the positions of the important genes and the functions of the gene clusters. The non-essential portion (primarily the b2 region) of the lambda genome can be replaced by a recombinant gene to establish a gene library. Scientists have carefully deleted most of the Lambdaestablish a gene library. Scientists have carefully deleted most of the Lambda genome, which can be used a vector for cloning a large segment of the genomic DNA and establish a gene library. Gene cloning and DNA analysis-An Introduction by

T.A. Brown, Blackwell Publishing – Chapter 2

Tools for Recombinant DNA TechnologyDNA Technology

The linear and the circular forms of Lambda DNA. (a) The linear form, showing the left and the right cohesive ends; (b) Base pairing between theends; (b) Base pairing between the cohesive ends results in the circular form of the molecule; (c) Rolling circle replication of produces a catenane of new lambda DNA molecules, which

i di id ll k d i t hare individually packaged into phage heads as new lambda particles are assembled. The length from one cossite to another is 49-52 kbp, which is very important for genome packaging y p g p g ginto lambda head. Less than 49 kbp or more than 52 kbp DNA will not package into a lambda head. This is regulated by an “A” protein located at the entry point of lambda headthe entry point of lambda head. Therefore for gene cloning and gene library purpose, it is necessary that the size of the recombinant gene is the same size as the deleted DNA on Lambda genome.


Documents

Separation of Proteins by gel electrophoresis · 2012-03-11 · All t DNA ith l d tAllow vector DNA with cloned gene to multiply Select clones by selectable marker(s) (antibiotic