My boss wants me to make a recombinant protein and this is something that I have never done before. The protein that I want to make is Recombinant Human Bone Morphogenetic Protein-7 and the product sheet of this compound where we first purchased the protein states that it is a "28.8 kDa homodimer, each subunit contains 116 amino acid residues (corresponding to amino acid residues 316 to 431 of the full-length BMP-7 precursor).
Can anyone recomend a protocol/manual/book/journal paper that describes the process of making this recombinant protein? Any help for this newbie would be greatly appreciated!
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#2
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Posted 14 May 2009 - 10:44 PM
This is a broad question I am afraid...
1) you need to know the sequence of your gene.
2) Once you have the sequence, you have decide what host to use for expression (E coli, Yeast, Insect cell?)
3) Then you choose appropriate vectors (eg: pET for E coli, pGAL for yeast... )
4) You can decide if you want fusion partner, eg 6xHIS tag, FLAG tag, GST tag etc
5) Then you start doing the cloning bits and transform into your host
6) Do expression tests
Ta
1) you need to know the sequence of your gene.
2) Once you have the sequence, you have decide what host to use for expression (E coli, Yeast, Insect cell?)
3) Then you choose appropriate vectors (eg: pET for E coli, pGAL for yeast... )
4) You can decide if you want fusion partner, eg 6xHIS tag, FLAG tag, GST tag etc
5) Then you start doing the cloning bits and transform into your host
6) Do expression tests
Ta
#3
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Posted 15 May 2009 - 04:55 AM
Gam8Doc, on May 13 2009, 05:03 PM, said:
Can anyone recomend a protocol/manual/book/journal paper that describes the process of making this recombinant protein? Any help for this newbie would be greatly appreciated!
Molecular Cloning: A Laboratory Manual (Sambrook)
http://www.amazon.com/Molecular-Cloning-La...3/dp/0879695773
An essential book for molecular biology labs
P
