Put the steps to produce spider silk protein in yeast in the correct order. 1 Purify protein. 2 Join yeast regulatory sequence and spider silk coding sequence by using enzymes. 3 Insert recombinant gene into yeast cells. 4 Use enzymes to remove yeast coding sequence. 5 Allow yeast to express spider silk gene.

The correct sequence is: 4>2>3>5>1.

Explanation:

The given question shows the sequence in which silk encoding gene from spider can be expressed in yeast in order to produce Recombinant Silk.Originally, silk can be obtained from spider. But for the commercial production and meeting the high demand of silk it is not economical to depend on spider to produce silk as they not only take long time to produce silk them but also they have a longer life-cycle and longer generation time as a result of which the quantity of silk obtained by spider per generation is very low. Also, spiders require specialized habitat and complex nutrient for their survival.Hence, yeast, an unicellular eukaryote, which has a shorter generation time and simple nutrient requirement for its survival, can be used for the commercial production of silk.For this, initially a yeast gene which is constitutively expressed in yeast is selected and the coding region of this gene is removed with the help of enzymes, keeping the regulatory sequences like the promoter, enhancer, terminator, etc, intact in the 5' and 3' region of the gene. A constitutive gene is the one which is expressed continuously at large amounts. This will result in the increased synthesis of the recombinant silk protein.Next, the coding sequence of the gene encoding for silk protein which is obtained from the spider is joined with the help of enzymes to the regulatory gene sequences of the yeast which was obtained in the first step.Next, this recombinant gene comprising of the yeast regulatory sequence and the coding region of the gene encoding for silk obtained from spider is inserted into the yeast cells.Next, the yeast cells are allowed to express the silk coding gene by the process of transciption and translation utilizing the yeast specific RNA (Ribonucleic acid) polymerase (recognizes the yeast-specific promoter and enhancer in the recombinant gene) and yeast-specific ribosome, respectively.The recombinant silk protein produced in the yeast can then be subjected to purification.

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