Experiment 9

Real time database teaching through internet (Bioinformatics)

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How to get gene information from

NCBI database
and construct a fusion protein

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outline
NCBI introduction How to use NCBI to get gene info:
Search paper , Nucleotide and protein Blast Alignment Analysis of Chromosome Location

Construction of fusion protein

Department of Biological Sciences and Biotechnology

NCBI introduction

NCBI introduction
National Center for Biotechnology Information

established on November 4, 1988, as a division of the

National Library of Medicine (NLM)

at the National Institutes of Health (NIH).

http://www.ncbi.nlm.nih.gov/

Department of Biological Sciences and Biotechnology

NCBI introduction

NCBI introduction

Department of Biological Sciences and Biotechnology

NCBI introduction

Department of Biological Sciences and Biotechnology

NCBI introduction

Department of Biological Sciences and Biotechnology

NCBI introduction

Department of Biological Sciences and Biotechnology

NCBI introduction

Department of Biological Sciences and Biotechnology

NCBI introduction

Department of Biological Sciences and Biotechnology

outline
NCBI introduction How to use NCBI to get gene info:
Search paper , Nucleotide and protein Blast Alignment Analysis of Chromosome Location

Construction of fusion protein

Department of Biological Sciences and Biotechnology

How to use NCBI

Search paper , Nucleotide and protein

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How to use NCBI

Search paper
Click onSearch
PubMed search paper Protein search protein Nucleotide search sequence Structure Genome Gene

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How to use NCBI

Search Nucleotide or protein

For example 1 : mouse acid phosphatase

Choose PubMed Input : mouse acid phosphatase

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How to use NCBI

For example 1 : mouse acid phosphatase

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How to use NCBI

For example 2 :protein of pincada fucata

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How to use NCBI

For example 2 :protein of pincada fucata

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How to use NCBI

For example 3download rice waxy gene

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How to use NCBI

For example 3download rice waxy gene

Department of Biological Sciences and Biotechnology

How to use NCBI

For example 3download rice waxy gene

Department of Biological Sciences and Biotechnology

How to use NCBI

For example 3download rice waxy gene

Department of Biological Sciences and Biotechnology

How to use NCBI

For example 3download rice waxy gene

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How to use NCBI

Blast

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How to use NCBI

Blast

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How to use NCBI

Blast

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How to use NCBI

Blast

Department of Biological Sciences and Biotechnology

How to use NCBI

Department of Biological Sciences and Biotechnology

How to use NCBI

Blast

Department of Biological Sciences and Biotechnology

How to use NCBI

Alignment
One of the cornerstones of modern bioinformatics is the comparison or alignment of protein sequences and DNA sequences. With the aid of multiple sequence alignments, biologists are able to study the sequence patterns conserved through evolution and the ancestral relationships between different organisms. Sequences can be aligned across their entire length (global alignment) or only in certain regions (local alignment). Common used software :clustal clustalw(http://www.ebi.ac.uk/clustalw/) Others: Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research, 2003, Vol. 31, No. 13. Bioedit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html

Department of Biological Sciences and Biotechnology

How to use NCBI

Department of Biological Sciences and Biotechnology

How to use NCBI

Department of Biological Sciences and Biotechnology

How to use NCBI

Department of Biological Sciences and Biotechnology

How to use NCBI

Analysis of Chromosome Location

Example 1 SMCX

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How to use NCBI

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How to use NCBI

Example 2 SMCX

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How to use NCBI

Department of Biological Sciences and Biotechnology

How to use NCBI

Department of Biological Sciences and Biotechnology

How to use NCBI

Department of Biological Sciences and Biotechnology

How to use NCBI

Department of Biological Sciences and Biotechnology

outline
NCBI introduction How to use NCBI to get gene info:
Search paper , Nucleotide and protein Blast Alignment Analysis of Chromosome Location

Construction of fusion protein

Department of Biological Sciences and Biotechnology

Construction of fusion protein

Construction of fusion protein

Fusion proteins, AKA chimeric proteins, are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. Chimeric mutant proteins occur naturally when a large-scale mutation, typically a chromosomal translocation, creates a novel coding sequence containing parts of the coding sequences from two different genes.

Department of Biological Sciences and Biotechnology

Construction of fusion protein

Recombinant fusion proteins

A recombinant fusion protein is a protein created through genetic engineering of a fusion gene. This typically involves removing the stop codon from a cDNA sequence coding for the first protein, then appending the cDNA sequence of the second protein in frame through ligation or overlap extension PCR. That DNA sequence will then be expressed by a cell as a single protein. The protein can be engineered to include the full sequence of both original proteins, or only a portion of either. This technique is often used for identification and purification of proteins, by fusing a GST protein, FLAG peptide, or a hexa-his peptide (aka: a 6xhis-tag) which can be isolated using nickel or cobalt resins (affinity chromatography).

Department of Biological Sciences and Biotechnology

Construction of fusion protein

Chimeric mutant proteins

Several drugs made from chimeric proteins are currently available for medical use. Several chimeric protein drugs are TNF blockers, such as Etanercept, Infliximab, and Adalimumab and immunosuppressants such as Daclizumab and Basiliximab.

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Construction of fusion protein

Prokaryotic expression system

In lab we commonly construct fusion expression plasmid with GST tag or His tag in E. coli BL21, induced with IPTG and finally purified the protein by the Ni column or the GST column. Commonly used Prokaryotic expression plasmid With His tag of pET series Novagen:http://www.emdbiosciences.com/g.asp?f=NVG/pE Ttable.html With the pGEX family of GST tagAmersham Biosciences

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Construction of fusion protein

pET-30 and pGEX series vector

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Construction of fusion protein

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Construction of fusion protein

Eukaryotic expression system

Construction of eukaryotic expression vector was mainly used to study gene expression in targeted cells, protein and protein interactions in cells, impact on cell growth, cell differentiation and apoptosis, as well as the construction of transgenic animals. The following is a list of three types of eukaryotic cell expression vector

pEGFPpcDAN3.1pCMV-Myc

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Construction of fusion protein

Department of Biological Sciences and Biotechnology

Construction of fusion protein

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Construction of fusion protein

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Construction of fusion protein

Key points
For both prokaryotic cell expression vector and eukaryotic cell expression vector

Select the appropriate expression vector; Choose protein coding sequences of the gene, that is, the "cDS" regional of genetic information "FEATURES" from NCBI

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Construction of fusion protein

According to the position of the fusion protein tag (such as His, GST) or other fusion protein (such as fluorescent protein), consider the fusion protein reading frame, design of appropriate primers (adding several bases to ensure a correct reading frame), connected appropriate restriction sites at both ends to ensure the correct translation of fusion protein;

Department of Biological Sciences and Biotechnology

Construction of fusion protein

After primer synthesis, PCR, restriction enzyme digestion, DNA recombination, and transformation process, finally recombinant plasmid will be gotten. Then transformed to appropriate cells for expression.

In E. coli, in accordance with the protocol, using appropriate conditions, induced the expression in E. coli BL21, and finally detecting the fusion protein; In eukaryotic cells, choose a suitable system according to the experiment purpose;

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Construction of fusion protein

When the fusion protein used for antibody, the fulllength target gene expression product can be chosen, and also the appropriate gene fragment (need to consider right section of the target gene to build fusion protein, the generally consider hydrophilicity or hydrophobicity and also epitope prediction from website)

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Thank you!
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