High-Throughput Cloning and Expression Library Creation for

Functional Proteomics

The International Proteomics Tutorial Program

What is cloning?

DNA cloningOrganism cloning

Cloning is a process where a genetically identical copy of an organism, cell or DNA is produced

Dolly sheep

DNA cloning and functional proteomics

DNA

Functional proteomics

Cell based assays Cell proliferationMorphologyDrug resistance

Protein functionInteraction partnersPTMs

Gene of interest Clone

Protein expressionin vivo or in vitro

Expression Libraries

Traditional Library DNA Clone Library

Expression library is the collection of clones capable to express protein. It stores genetic information to facilitate the study of the roles of genes and proteins in a systematic and high-throughput (HTP) manner.

Main enzymes used for DNA cloning

(1) DNA polymerases

(2) Restriction enzymes and ligases

(3) Recombinases

DNA polymerase

DNA polymerase can use RNA or DNA as template.

DNA dependent-DNA polymerase copy the DNA and can be used for DNA amplification in vitro in a Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction

DNA polymerase

DNA polymerase can use RNA or DNA as template.

DNA dependent-DNA polymerase copy the DNA and can be used for DNA amplification in vitro in a Polymerase Chain Reaction (PCR)

RNA dependent-DNA polymerase is used to copy the information from the RNA to the DNA. The new DNA molecule is name copy DNA or cDNA

cDNA synthesis

Type II Restriction Enzyme and Ligase

Type II Restriction enzymes – “scissors” capable of identifying and cleaving specific DNA sequences

Ligase - re-join two DNA strands

Recombinases

Recombinases can cut and ligate DNA fragments, within a single complex

Bidirectional recombination

Recombinases

Unidirectional recombination

Recombinases can cut and ligate DNA fragments, within a single complex

Steps for DNA cloning

1) Identification of appropriate insert and insert source

2) Selection of appropriate vector

3) Selection of appropriate cloning strategy

Insert selection - What is the structure of the gene in the genome?

Prokaryotic RNA5’UTR Coding sequence(CDS)Start Stop 3’UTR

Open Reading Frame (ORF)

Prokaryotic genomic DNACoding sequence(CDS)

Transcription

Eukaryotic genomic DNA

Mature eukaryotic mRNA

Pre-mRNA

5’ 3’

Cap 5’UTR Coding sequence(CDS) 3’UTR Poly-A tailStart Stop

3’

Capping, splicing, polyadenylation

Open Reading Frame (ORF)

5’

5’UTR 3’UTRExon ExonExon

Intron IntronIntron

Exon

5’

5’UTR 3’UTRExon ExonExon

Intron IntronIntron

Exon

Insert selection - What is the structure of the gene in the genome?

ORFs – The insert of choice for expression clones

Due to the lack of introns, ORFs can be expressed to study protein functions.

Templates to Generate ORF Clones

(1) Genomic DNA: all genes equally present but not suitable for genes with introns (most eukaryotes);

(2) cDNA: no introns between exons but the biases on mRNA expression exist both temporary and spatially;

(3) Vector with the gene of interest (GOI): gene specific primers can be designed to amplify the GOI for the cloning into the desired vectors;

(4) Gene Synthesis (Chemical or PCR-based method). Allows the codon usage optimization. High cost.

Templates to generate

ORF clones

Vectors

Entry vector common features: • ori (origin of replication)• selection marker • cloning site

Inserts cloned into entry vectors are NOT expressed

Expression vectors

Additional features in the expression vector : • promoter (required) • tag (optional)

Necessary characteristics of high-throughput cloning systems

• Properties of the enzymes used for cloning

• Fidelity

• Efficiency

• Stability

• Clone validation

• Automation Friendly

• Cost

Example of available high-throughput cloning systems

1. Restriction Enzyme Based Cloning(1) Flexi® Vector Systems (Promega)

2. Recombination Based Cloning(1) Creator Cloning System (Cre-loxP, Clontech)

(2) Gateway® Technology (phage lambda – attB/P, Life

Technologies)

Flexi® vector systems• Flexi cloning system is a restriction enzyme-based cloning system that uses two rare-cutting restriction enzymes, SgfI and PmeI

• Flexi vectors carry a lethal gene in between SgfI and PmeI to allow the efficient cloning selection.

Frequency of cDNAs or open reading frames(ORFs) lacking SgfI or PmeI sites

Flexi® Vector Systems Technical Manual@Promega

Creating Flexi® clones

Lethal gene in the expression vectors provides negative selections for the parent vectors

Generation of Flexi clones with amino-terminal tag

5’…GTTTAAAC…3’3’…CAAATTTG…5’

PmeI sequence:PmeI sequence:

•Reconstitution of SgfI site encodes Ala-Ile-Ala and PmeI site encodes Val-Stop-Thr.• GOI will be translated from the tag’s start codon to the stop codon present in the gene or the PmeI site (in red);

SgfI sequence:SgfI sequence:

5’…GCGATCGC…3’3’…CGCTAGCG…5’

+Digestion,

ligation and

selection

5’…GTTTAAAC…3’3’…CAAATTTG…5’

5’…GAGCTC…3’3’…CTCGAG…5’

PmeI PmeI EcolCRIEcolCRI

5’…GTTTCTCNN…3’3’…CAAAGAGNN…5’

New SequenceNew Sequence

Ligation

• Ligation of PmeI and EcolCRI sites disrupt both PmeI and EcolCRI sites, new sequence encodes Val-Phe-X, X determined by GNN;• It is inadvisable to transfer GOI into C-terminus Flexi vectors if you plan to transfer it into other Flexi vectors in the future.

Generation of Flexi clones with carboxyl-terminal tag

+Digestion,

ligation and

selection

Transferring genes between distinct Flexi vectors Acceptor vector with amino-terminal tag

SgfI & PmeI digestion

LigationTransformation

Antibiotic 2 selection

SgfI & PmeI digestion

SgfI & EcolCRI digestion

LigationTransformation

Antibiotic 2 selection

Transferring genes between distinct Flexi vectors Acceptor vector with carboxyl-terminal tag

+ +

Creator cloning system

•Creating Master Clones With In-Fusion: In-Fusion enzyme fuses DNA fragments, e.g., PCR generated sequences and linearized vectors, precisely by recognizing a 15bp overlap at their ends.

•Creating Expression Clones With Creator Cloning: 1. Cre recombinase mediates the recombination between DNA sequences at loxP sites; 2. Donor vectors contain two loxP sites, which flank the 5’ of GOI and 5’ of CmR resistance gene; 3. Acceptor vectors contain a single loxP site, followed by a bacteria promoter to facilitate the expression CmR gene after recombination.

LoxP sequences:LoxP sequences:

In-Fusion® HD Cloning Kit User Manual@Clontech

Creating entry clones with In-Fusion

Creating expression clones with Creator cloning system

Tags in the Creator expression vectorsN-terminal Tag

Tags in the Creator expression vectorsC-terminal Tag

Gateway cloning system

•Creating Gateway Master Clones Using BP reactions BP clonase recombine linear PCR product with entry vectors.

•Creating Gateway Expression Clones Using LR reactions LR clonase recombine entry clone with expression vectors.

5’UTR 3’UTR

Gene of Interest(GOI)

Generation of PCR fragment with attB sites

Gene of Interest(GOI)

1st PCR using gene specific primers + Partial attB1/attB2 site sequences

2nd PCR using universal primers including the full length attB1/attB2 site sequences

attB1 attB2

Gene of Interest(GOI)

•attB1/attB2 site sequences are adapted from phage λ that allows the site specific recombination between DNA fragments;•Two rounds of PCR are applied for the addition of adaptor sequences to avoid potential mutations in the synthesis of long primers.

Creating Gateway entry clones using BP reactions

Creating Gateway expression clones using LR reactions

Tags in the Gateway expression vectorsN-terminal Tag

Tags in the Gateway expression vectorsC-terminal Tag

Generating new expression vectors

Flexi Cassette

Gateway Cassette

Creator Cassette

Overall principle: Insert the required cloning cassette into the desired expression vectors by any cloning

method (restriction enzyme, In-Fusion).

Alternative cloning methodsIn-Fusion

Alternative cloning methodsGibson Assembly

Alternative cloning methodsLigation Independent Cloning

Alternative cloning methodsGold Gate Assembly

Alternative cloning methodsUnivector Plasmid Fusion System

Comparison of distinct cloning systemsFlexi Creator Gateway

Cloning steps

8 s teps (PCR, digestion, DNA purifi cation, anneal ing,

transformation, colony picking, DNA extraction, sequencing)

12 s teps (PCR, vector digestion, DNA purifi cation/cloning enhance, In-Fus ion, transformation, colony

picking, DNA extraction, sequencing, Cre-reaction,

transformation, colony picking, DNA extraction, clone va l idation )

12 s teps (PCR1+PCR2 - s ingle tube reaction, DNA purifi cation, BP

reaction, transformation, colony picking, DNA extraction, sequencing, LR-reaction,

transformation, colony picking, DNA extraction, clone va l idation )

Dependent

If BP reaction is employed the gene size should be <3Kb. Genes larger than

3Kb can be cloned using other approaches

Dependent

Genes with either SgfI or PmeI sites should be cloned using alternative

protocols

Necessary Necessary

Alternative: use lox P site (34 nt) as adapter sequence in the PCR reaction

Alternatives: use attL site (100 nt) as adapter sequence in the PCR reaction; or a expression vector with attP sites, which will add 100 nt on each side of

the ORF

Primer lengh (adapter sequence only)

8 nt (restriction enzyme recognition s i te)

15 nt (vector extremity sequence) or 34nt (loxP sequence; cloning

di rectly to the express ion vector)

25 nt (attB s i te) or 42nt (attB s i te + Shine-Dalgarno sequence + Kozak sequence) for the express ion of

native protein

Independent

Size of cloned genes Any Any

Sequence of the gene of interest

Independent

Entry vector Not necessary

Flexi Creator Gateway

N-terminal : 3 N-terminal : 12 N-terminal : 9

C-terminal : 1C-terminal : 0

(loxP s i te i s removed by spl icing) C-terminal : 9

Amino-terminal tags (closed ORFs)

Express ion of the SgfI s i te. Addition of 3 aa between the tag

and the GOI

Express ion of the loxP s i te. Addition of 12 aa between the tag

and the GOI.

Express ion of the attB1 s i te. Addition of 9 aa between the tag

and the GOI.

Carboxyl-terminal tags (native start codon)

Express ion of the EcoICRI s i te. Addition of 3 aa between the tag

and the GOI

Express ion of C-termina l tag from the express ion vector only i f the

express ion systems is able to perform RNA spl icing

Express ion of the attB2 s i te. Addition of 9 aa between the tag

and the GOI

Ligation – Medium In-Fus ion – LowBP reaction – Low (<3Kb genes),

High for larger genes

Cre reaction - High LR reaction – Low

Bacterial competency needed (cfu)

10^6 10^8 10^6

Number of expression vectors compatible with

the system

+ ++ ++++

Number of clones libraries compatible with the

system

+ ++ ++++

Cost per construct (enzymes only)

$4.80 - $21.02 (depending on the vendor)

$14.00 - $28.10 (In-Fus ion - $12.98, Cre - $1.02-

15.12)

$16.33 (BP mix - $7.99, LR mix - $8.34)

Potential number of additional amino acids

introduced via the cloning site

Number of false positives

Proteomics application of expression clone libraries

Generation of protein microarray with 2000 distinct ORFs from Vibrio cholerae for

the identification of immune response in patients with Cholera

Adapted from Festa et al, 2012

DNA

Protein