Making enzyme

[demymahi]

Hi everyone

I am new to Molecular Biology and it is not my field either.
But I have recently started reading about it and found it very interesting.
I have a few basic questions.
Can any one please explain me how enzyme making work?
I mean how you can make your enzyme? If someone can explain me step by step, that will be great. What is the difference making enzyme and recombinant enzyme?

[mchlbrmn]

I think there are two general ways one could produce enzymes.
The first is to purify it out of the biological material it naturally occurs in. For example, to produce a heat tolerant enzyme one might isolate an organism that lives in hot springs, and then grind it up and go through various steps to purify the enzyme from the other constituents present.
The recombinant alternative, as you mentioned, is to clone the DNA of the gene that codes for the enzyme in this organism, and put it into a vector that can express the gene in bacteria or yeast. A bacterial system has the advantages that bacteria grow extremely rapidly, they might produce the protein in huge amounts, and a tag might be added to the end of the protein that can be used to fish it out later to make purification easier.

[relaxin]

Enzymes function as catalysts, which means that they speed up the rate at which metabolic processes and reactions occur in living organisms. Enzymes are mostly proteins, but can be RNA (ribozymes).

Besides cloning and expression of cDNA encoding an enzyme from a natural source, now it is possible to chemically synthesize the cDNA encoding the enzyme using optimized codons for the species you are working with. Direct protein synthesis of the enzyme will be still to expensive. Of course, you can easily synthesize ribosymes.

[demymahi]

Thank you very much for your reply and time.
So first step would be to get a gene which need to be sequenced right? Hope I have understood this right,
1) get gene
2)clone this gene into organism
3)put it into a vector
4) purified the gene

then what you do to make recombinant?

Or can you suggest me some really good book or website where I can read this?

Thanks once again

[CrowSan]

Get gene
Put gene into vector (to infect/transform another organism)
Put gene-vector into organism
The last step (step 4) is purify/harvest the protein the gene codes for.

When you put a "foreign" gene into an organism it does not come from it is automatically a "recombinant" gene.
Wikpedia (as always) has the answers:
http://en.wikipedia.org/wiki/Recombinant_DNA

[relaxin]

1. Get gene
You can copy it by RT-PCR, if you know the sequence. Alternatively, you can ask someone who has the cDNA, or purchase from a company.

2. Put gene into expression plasmid
There are three main types of expression systems; bacterial, insect and mammalian. The choice depends on your objective. Some systems can be inducible, some have built-in tag for detection or purification.

3. Transfect or transform the corresponding host with the expression plasmid
After transfection or transformation, you need to select with antibiotic for cells harboring the expression plasmid. You can then induce for protein expression.

4. Purify the recombinant protein
Various built-in tags will facilitate the purification of recombinant protein. For the bacterial expression system, the protein produced is often insoluble. Purification of recombinant protein is often done under denaturing conditions. The purified recombinant protein will require protein refolding before enzymatic activity can be determined.