FSH and LH are synthesized and secreted by the same type of cells in anterior pituitary. Both the hormones are heterodimers composed of a common a-subunit linked to distinct b-subunit, both the subunits contain oligosaccharide chains. There are multiple sugar moieties that are highly complex in nature and are essential for biological activity. Owing to their similar biochemical properties and the presence of multiple isoforms with heterogeneous oligosaccharide moieties, it has been rather difficult to obtain pure preparations of FSH or LH. Preparations of FSH extracted from the pituitary contain small amounts of contaminating hormones such as LH, which may produce unexpected activity, as a result of the synergistic action of FSH and LH, in studies designed to investigate FSH activity, resulting in unclear results.

            Recombinant FSH is guaranteed to be pure and free from cross-contaminating hormones. Several workers have expressed FSH in Chinese hamster ovary cells. Although mammalian cells can add complex oligosaccharide chains and form correct intramolecular disulphide bonds that are essential for FSH and LH activities, the yield of the recombinant molecules from mammalian cell expression system are generally lower than those achieved by bacteria, yeast or insect cell systems. We have been successfully produced bioactive r-oFSH by baculovirus expression vector system and has also achieved the partial purification of the expressed protein. There are indirect evidences to suggest that r-protein has correct annealing of the two subunits and proper folding and glycosylation of the hormone.

·    Two expression cassettes, pVJ-5a and pVJ-7b, have been constructed, one has the a subunit gene and the other has b subunit gene in right orientation under the strong polyhedrin promoter.

·     Co- transfection of Sf-9 cells with both these constructs resulted in expression of two genes two genes and yielded immunoreactive r-FSH, suggesting that correct dimerization and secretion of the hormone was carried out successfully in-vivo.

·     Another construct, pVJMP-23 has been made which contains the entire coding regions of both the a and FSHb subunit genes in the right orientation in the same plasmid but under separate P_polh promoters.

·     Transfection of Sf-9 insect cells with pVJMP-23 resulted in the successful production and secretion of r-FSH in more efficient manner.

·      The expression of r-FSH has been achieved by both single and dual promoter vectors. Quantification by RIA shows that the amount of FSH produced is 12 mIU and 24 mIU / million cells respectively. This shows that it is advantageous to clone both the subunits in a single plasmid

·     In our efforts to search for optimal system that can provide high level expression, yet another strategy has been used to express both the a and b subunit genes. A construct pVJIRES-32 has been made that contains both the a and b subunit genes in the right orientation under the same CMV promoter but separated by an IRES fragment. It also contains neomycin gene which provides resistance to G 418 and serves as a selection marker

·     Expression of pVJIRES-32 and production of r-FSH has been achieved in CHO cells. Analysis of the culture supernatant reveals the presence of desired protein. Quantification by RIA that the amount of FSH produced in 12.5 mIU / million cells / 48 h

·     The bioactivity of r-FSH studied in an in vivo Steelman and Pohley  assay  showed that there was a significant increase in the weight of the reproductive organs, hence proving that r-FSH produced is biologically active and is able to stimulate the growth and proliferation of ovaries and uteri in immature mice.

·     SDS-PAGE analysis of the secreted protein showed the desired band at 28kDa. The molecular weight analysis confirmed that the configuration of r-FSH was similar to the natural hormone and the two subunits made by this route are capable of annealing with each other

·     Partial purification of r-FSH was achieved by a combination of ammonium sulphate precipitation and affinity chromatography on Con-A sepharose columns. Protein was eluted from the column in four fractions. Maximum activity of FSH as analysed by RIA was found in the peak eluted with 0.2 M MeMan.

·     SDS-PAGE analyses showed the desired band of 28kDa in the second peak eluted with 0.2M MeMan. This band was absent in flowthrough indicating that all the protein bound to the lectin column.

·     Administration of partially purified r-FSH into immature mice resulted in substantial increase in the weight of the reproductive organs, which showed that no activity of the r-FSH was lost during purification procedures.

·     These data provide ample evidence that correct annealing and dimerisation of a and b subunits have taken place and the r-FSH is correctly folded and properly glycosylated. The biological activity has been found to be substantially high which proves the feasibility of insect cells to synthesize complex glycoproteins such as r-FSH.