Question

What two restriction enzymes could you use if you wanted to produce a protein that was fused to a GST-tag that could be removed using thrombin? Would this experimental design place any other tags on your protein?

Here is the vector:

T7 promoter lac operator Xbal rbs Ndel AATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGTCCCCT Met Ser Pro GST Ta His TagSacl ATACTAGGTTAT.627bp...GACCATCCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTCATCACCATCACCATCACTCCGCGGGTCTGGTGCCACGCGGTAGT lle Leu Gly Tyr.. .209aa. . . Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser Gly Ser Gly His His His His His His Ser Ala Gly Leu Val Pro Arg Gly Ser thrombin S-Tag primer #69945-3 s-та Munl PshAl ACTGCAATTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGGTGGTGGCTCCGGTGATGACGACGACAAG Thr Ala lle Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg GIn His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly Asp Asp Asp Asp Lys enterokinase Pstl Eagl Sac Sal HindIlI Notl AscI Sse8387 His Ta Ncol ECOR V BanHI EcoRI BsrGI Stul Xhol AGTCCCATGGGATATCGGGGATCCGAATTCTGTACAGGCCTTGGCGCGCCTGCAGGCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACCACCACTAATTGATTAA Ser Pro Met Gly Tyr Arg Gly Ser Glu Phe Cys Thr Gly Leu Gly Ala Pro Ala Gly Glu Leu Arg Arg GIn Ala Cys Gly Arg Thr Arg Ala Pro Pro Pro Pro Pro Pro Pro Leu Ile Asp End pET-41a(+) AGTCCCATGGATATCGGGGATCCGAATTCTGTACAGGCCTTGGCGCGCCTGCAGGCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACCACCACTAATTGATTAA pET-41b(+) Ser Pro Met Asp Ile Gly Asp Pro Asn Ser Val Gin Ala Leu Ala Arg Leu Gin Ala Ser Ser Val Asp Lys Leu Ala Ala Ala Leu Glu His His His His His His His His End AGTCCCATGGCGATATCGGGGATCCGAATTCTGTACAGGCCTTGGCGCGCCTGCAGGCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACCACCACTAATTGATTAA pET-41c(+) Ser Pro Met Ala le Ser Gly lle Arg lle Leu Tyr Arg Pro Trp Arg Ala Cys Arg Arg Ala Pro Ser Thr Ser Leu Arg Pro His Ser Ser Thr Thr Thr Thr Thr Thr Thr Thr Asn End Avril pu1102 TACCTAGGCTGCTAAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG T7 terminator T7 terminator primer #69337-3 pET-41a(+) cloning/expression regions

Answer 1

The gene of interest can be cloned in the restriction sites :Mun I and Xho I, to produce a protein that is fused to GST-tag at the N terminal, with a thrombin cleavage site in between of them. This stratergy will also put a His tag, both at the N and C terminal of the recombinant protein.