Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

Mammalian protein expression system has the function of protein folding and post-translational modification which let the protein closer to the natural protein, so that to obtain the same biological activity with natural protein. Therefore, the Mammalian Cell Expression System is the most widely used in the development and production of recombinant protein drugs, particularly in the therapeutic monoclonal antibodies. Email:marketing@medicilon.com.cn web:www.medicilon.com

The production of mammalian proteins in suYcient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a signiWcant number of mammalian proteins. This creates the necessity of well-identiWed expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the Xuorescent protein EGFP as a model, it is shown that muscle Wbers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of »1 mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement. A general problem in biology is the design of protein expression systems able to express authentic mammalian proteins in large enough quantities to satisfy the necessities of various biomedical applications. Prokaryotic expression systems, though powerful in their ability to generate massive quantities of recombinant proteins, have severe limitations for the expression of properly folded (and processed) fulllength replicas of a large number of eukaryotic proteins. Although these limitations pertain to both cytosolic and membrane proteins, they have become a critical deWciency for the study of integral membrane proteins such as ion channels and transporters. Thus, alternative expression systems able to approximate the yield of bacteria, but that overcome some of their limitations, have been developed. Of these, yeast and baculovirus expression systems have been considered favorable hosts for the expression of foreign eukaryotic proteins for research, industrial or medical use. However, since biological activity depends critically on the proper folding and authenticity of proteins, their expression in bona Wde mammalian systems has become a necessity. Cultured mammalian cells have been utilized as expression systems to fulWll this role, but issues related to the creation of stable cell lines, their relatively low yield, and the requirement of balanced delicate nutrients weaken their eVective use for large-scale protein production. Important advances in gene manipulation, plasmid design, and in vivo transfection methodologies during the past decade have led to the proposal that skeletal muscles can be used as a potential factory of proteins for gene therapy. In this paper, we explore the alternative use of mammalian skeletal muscle as a transient expression system for mass production of eukaryotic heterologous proteins. Salient features that make skeletal muscle amenable for this purpose are that: they represent a large proportion of the body mass; they are easily accessible for transfection procedures; and, they are composed of large syncytial postmitotic cells with large capacity to synthesize proteins . However, up until now, most experimental evidence seems to suggest that skeletal muscle tissue may be unable to synthesize recombinant proteins at a rate, and in quantities (on a scale of mg of protein/g wet weight of tissue), compatible with its potential use as an eVective protein expression system. This paper aims to dispel these qualms. To this end, we used: (a) in vivo electroporation as the method of choice to deliver controlled amounts of plasmids into well-identiWed limb muscles of the mouse; (b) pDNA vectors to encode for a genetically engineered derivative of the green Xuorescent protein that have enhanced Xuorescence in the green (EGFP) region of the visible spectrum as an example of a medium sized (27 kDa) transgenic cytosolic protein; (c) two-photon laser scanning confocal Xuorescence microscopy (TPLSCM)1 to monitor the expression of Xuorescent proteins in live tissue at the organ (whole muscles), cellular (single muscle Wbers), and sub-cellular (intra-sarcomeric distribution) levels; and Wnally, (d) standard biochemical techniques to isolate and characterize the Xuorescent proteins synthesized by muscle tissue, and spectroXuorimetry to quantitate the expression yield. Altogether, our data demonstrate that mammalian skeletal muscle is a strong heterologous protein expression system, which due to the simplicity of its use may become an important biological tool in the future. Materials and methods Animal model Male C57BL mice 1.5–3 months of age, weighting 20– 30 g, were used. Plasmid transfection was performed in either FDB muscles or a group of “lower limb muscles” which predominantly included the soleus, tibialis anterior, and extensor digitorum muscles. Experiments were carried out according to the guidelines laid down by the UCLA Animal Care Committee. Plasmids ampliWcation The plasmid encoding for EGFP (pEGFP-N2) was obtained from Clontech. It was ampliWed in OneShot TOP10 (Clontech) bacteria and were isolated using Qiagen Endo-Free Kits (Qiagen, Valencia, CA, USA) following the procedures of the manufacturer. Muscle transfection with DNA plasmids Muscle transfection was achieved by injection with pDNA, followed by in vivo electroporation, in anesthetized animals (isoXurane). The protocols used in FDB muscles diVered slightly from those in “lower limb” muscles. In the case of FDB, 5 l of 2 mg/ml hyaluronidase was dissolved in sterile saline and injected subcutaneously into the foot pads of the animal using a 33-gauge needle. An hour later, »20 g of pDNA, dissolved in buVer (10 mM Tris–Cl, pH 8, 1 mM EDTA) at concentrations of 2–5 g/l, was injected. After 10 min, two electrodes (200 m gold plated stainless steel needles) were placed subcutaneously close to the proximal and distal tendons of the muscles in order to deliver electrical pulses for muscle electroporation. The pulse protocol was: 20 pulses of 100 V in amplitude, 20 ms in duration, applied at a frequency of 1 Hz. Pulses were generated by a Grass S88 medical stimulator (Grass, Quincy, MA, USA). For lower limb muscles, the protocol was the same except that the injections of hyaluronidase and pDNA were applied intramuscularly at three positions (equidistant locations between the ankle and the knee) of the lower limb muscles. Stimulating electrodes were placed parallel to the leg axis and inserted subcutaneously at both sides of the leg. In general, right muscles were transfected while left muscles were used as contra-lateral controls.