Expression problem - (Mar/29/2008 )

My fellow has cloned alpha amylase gene of bacillus licheniformis in E. coli (DH5 ά) using pTZ57R/T (first cloning step) and then in E. coli BL21 using PeT-21 (second cloning step). Now i have to proceed further. After taking expression in BL21 and SDS analysis expression is visible. But protein is in form of inclusion bodies. as there was very less enzyme activity after sonication. But what i was told that tranformed cells of E. coli BL21 are stable only for 2-3 weeks and after that I have to prepare competent cells of BL21 and again tranformed it with recombinant plasmid. I could nt find it out why it is so. Moreover, Tranformed cells of BL21 if are preserved in glycerol stock there are very less chances for expression even though the might contain the recombinent plasmid.
1. Is there any possible way by which i can maintain/preserve tranformed cell of BL 21 with expression
2. Second I need protocol for solubilzation of recombinant protein.

If any one could help. I 'd be gr8ful.

Regards

I use BL21(DE3) more than BL21 but from what I know the only difference is method of expression. That said, my transformed BL21(DE3)s are only good for expression if they're on a plate for less than 3 days. I keep a 25% glycerol stock in the -80 to streak out as needed and have not had any problems with expression. Test it out for yourself and see how well it works. You won't lose anything by making a gly stock, especially since, if it doesn't work you'll have to retransform anyways.

To purify proteins in inclusion bodies, search for a denaturing (his tagged) protein prep. It starts out similarly to a soluble prep, but you'll be working with the pellet in a guanidine or urea buffer after the first clarifying spin.

do you want your protein to be in native or denatured form? Judes method to get denatured protein might works but it can be hassling to renature it again. But then again, to solubilise inclusion bodies, denaturant chemicals are the best to do it.

how about getting those commercial lysing kit? apparently, some claimed that it even solubilise inclusion bodies in native form. try sigma. i used that before and it works well without the use of sonicator.

good luck.

Thanks

But i want proteins in active form not denatured and kit method is nt possible.

active form. Some kits can be used to have active protein.

renaturing is an option too.

Try slowing down the expression rate. Reduce the expression temperature to 25 or even 18 C. You'll need to extend the expression time before you harvest.
Also, you could try reducing the amount of IPTG you add. I would suggest you try a couple of amounts, say, 1/5 and 1/10 concentration of your original amount.

You might also need to check the codon usage of your gene. There are a number of websites that do this, including http://nihserver.mbi.ucla.edu/RACC . Enter your sequence, and the software will tell you if you have any rare codons. Rare codons near the N-terminus are bad, runs of them are very bad. You might have to consider using an E. coli strain like Rosetta, which contains some extra tRNA genes.

Thanks swanny you have given really useful suggestions. I will follow these. I have checked the rare codon in gene these are present here but how to determine that if these are present near N-terminus. would you please guide me about it here is the gene sequence of alpha amylase.

1 ATGAAACAAC AAAAACGGCT TTACGCCCGATTGCTGACGC TGTTATTTGC GCTCATCTTC
61 TTGCTGCCTCATTCTGCAGCAGCGGCGGCAAATCTTAATG GGACGCTGATGCAGTATTTT
121 GAATGGTACATGCCCAATGACGGCCAACATTGGAAGCGCTTGCAAAACGACTCGGCATAT
181 TTGGCTGAACACGGTATTACTGCCGTCTGGATTCCCCCGG AACGAGCCAA
241 GCGGATGTGGGCTACGGTGCTTACGACCTT TATGATTTAGGGGAGTTTCATCAAAAAGGG
301 ACGGTTCGGACAAAGTACGGCACAAAAGGAGAGCTGCAATCTGCGATCAA AAGTCTTCAT
361 TCCCGCGACATTAACGTTTACGGGGATGTGGTCATCAACCACAAAGGCGGCGCTGATGCG
421 ACCGAAGATGTAACCGCGGTTGAAGTCGATCCCGCTGACCGCAACCGCGTAATTTCAGGA
481 GAACACCGAATTAAAGCCTGGACACATTTTCATTTTCCGGGGCGCGGCAGCACATACAGC
541 GATTTTAAAT GGCATTGGTACCATTTTGACGGAACCGATTGGGACGAGTCCCGAAAGCTG
601 AACCGCATCTATAAGTTTCAAGGAAAGGCT TGGGATTGGGAAGTTTCCAATGAAAACGGC
661 AACTATGATTATTTGATGTATGCCGACATCGATTATGACCATCCTGATGTCGCAGCAGAA
721 ATTAAGAGATGGGGCACTTGGTATGCCAATGAACTGCAATTGGACGGTTTCCGTCTTGAT
781 GCTGTCAAACACATTAAATT TTCTTTTTTGCGGGATTGGGTTAATCATGTCAGGGAAAAA
841 ACGGGGAAGGAAATGTTTACGGTAGCTGAA TATTGGCAGAATGACTTGGG CGCGCTGGAA
901 AACTATTTGA ACAAAACAAA TTTTAATCAT TCAGTGTTTGACGTGCCGCT TCATTATCAG
961 TTCCATGCTGCATCGACACAGGGAGGCGGCTATGATATGAGGAAATTGCT GAACAGTACG
1021 GTCGTTTCCA AGCATCCGTTGAAAGCGGTT ACATTTGTTGATAACCATGATACACAGCCG
1081 GGGCAATCGCTTGAGTCGACTGTCCAAACATGGTTTAAGCCGCTTGCTTACGCTTTTATT
1141 CTCACAAGGGAATCTGGATACCCTCAGGTT TTCTACGGGGATATGTACGG GACGAAAGGA
1201 GACTCCCAGC GCGAAATTCC TGCCTTGAAACACAAAATTGAACCGATCTTAAAAGCGAGA
1261 AAACAGTATGCGTACGGAGCACAGCATGATTATTTCGACC ACCATGACATTGTCGGCTGG
1321 ACAAGGGAAGGCGACAGCTCGGTTGCAAATTCAGGTTTGGCGGCATTAATAACAGACGGA
1381 CCCGGTGGGGCAAAGCGAATGTATGTCGGCCGGCAAAACGCCGGTGAGAC ATGGCATGAC
1441 ATTACCGGAA ACCGTTCGGA GCCGGTTGTCATCAATTCGGAAGGCTGGGG AGAGTTTCAC
1501 GTAAACGGCG GGTCGGTTTC AATTTATGTT CAAAGATAG

Regards