Cloning and expression of recombinant human GMCSF from Pichia pastoris GS115--a progressive strategy for economic production.

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is a proinflammatory cytokine and hematopoietic growth factor. Recombinant human granulocyte-macrophage colony-stimulating factor (rhGMCSF) serves as a biotherapeutic agent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. The objective of the present study includes construction of rhGMCSF having N-terminal intein tag, expression of protein both extracellularly and intracellularly from yeast expression system followed by its purification in a single step by affinity chromatography. The soluble and biologically active rhGMCSF was obtained from Pichia pastoris GS115. About 122 g DCW/L of final yield was obtained for both cytosolic and secretory expression of Pichia GS115 strain. Purified intracellular hGMCSF was 420 mg/L with a specific activity of 2.1×108 IU/mg, and the purified extracellular recombinant protein was 360 mg/L with a specific activity of 1.9×108 IU/mg. The data presented here indicate the possibilities of exploring the economic ways of producing the rhGMCSF.

Utility of multi-channel surface electromyography in assessment of focal hand dystonia.

INTRODUCTION: Surface electromyography (SEMG) allows objective assessment and guides selection of appropriate treatment in focal hand dystonia (FHD). METHODS: Sixteen-channel SEMG obtained during different phases of a writing task was used to study timing, activation patterns, and spread of muscle contractions in FHD compared with normal controls. Customized software was developed to acquire and analyze EMG signals. RESULTS: SEMG of FHD subjects (20) showed "early onset" during motor imagery, rapid proximal muscle recruitment, agonist-antagonist co-contraction involving proximal muscle groups, "delayed offset" after stopping writing, higher rectified mean amplitudes, and mirror activity in contralateral limb compared with controls (16). Muscle activation latencies were heterogenous in FHD. CONCLUSIONS: Anticipation, delayed relaxation, and mirror EMG activation were noted in FHD. A clear pattern of muscle activation cannot be ascertained. Multi-channel SEMG can aid in objective assessment of temporal-spatial distribution of activity and can refine targeted therapies like chemodenervation and biofeedback.

Single step intein-mediated purification of hGMCSF expressed in salt-inducible E. coli.

Human granulocyte-macrophage colony stimulating factor (hGMCSF) is an important therapeutic cytokine. As a novel attempt to purify hGMCSF protein, without the enzymatic cleavage of the affinity tag, an intein-based system was used. The gene was fused by overlap extension PCR to the intein sequence at its N-terminal in pTYB11 vector. The hGMCSF was expressed as a fusion protein in E. coli BL21(DE3), and E. coli GJ1158. In the former, the protein was expressed as inclusion bodies and upon purification the yield was 7 mg/l with a specific activity of 0.5 x 10(7) IU/mg. In salt-inducible E. coli GJ1158, hGMCSF was expressed in a soluble form at 20 mg/l and a specific activity of 0.9 x 10(7) IU/mg. The intein-hGMCSF was purified on a chitin affinity column by cleaving intein with 50 mM DTT resulting in a highly pure 14.7 kDa hGMCSF.

Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris.

As a novel attempt for the intracellular recombinant protein over expression and easy purification from Pichia pastoris, the therapeutic cytokine human granulocyte macrophage colony stimulating factor (hGMCSF) gene was fused to an intein-chitin-binding domain (gene from pTYB11 vector) fusion tag by overlap extension PCR and inserted into pPICZB vector, allowing for the purification of a native recombinant protein without the need for enzymatic cleavage. The fusion protein under the AOX1 promoter was integrated into the P. pastoris genome (SMD 1168) and the recombinant Pichia clones were screened for multicopy integrants. Expression of hGMCSF was done using glycerol and methanol based synthetic medium by three stage cultivation in a bioreactor. Purification of the expressed hGMCSF fusion protein was done after cell disruption and binding of the solubilized fusion protein to chitin affinity column, followed by DTT induced on column cleavage of hGMCSF from the intein tag. In this study, final biomass of 89 g dry cell weight/l and purified hGMCSF of 120 mg/l having a specific activity of 0.657 x 10(7) IU/mg was obtained. This strategy has an edge over the other--His or--GST based fusion protein purification where non-specific protein binding, expensive enzymatic cleavage and further purification of the enzyme is required. It distinguishes itself from all other purification systems by its ability to purify, in a single chromatographic step.

Motor and somatosensory evoked potentials in a primate model of experimental spinal cord injury.

Motor and somatosensory evoked potentials (MEP and SSEP) were compared after experimental spinal cord injury in Bonnet monkeys (macaca radiata). The MEP and SSEP changes following graded injuries were related to clinical outcome. Eight healthy mature monkeys with a mean weight of 4.2 + 0.9 Kg were chosen for the study. Graded spinal cord injury was caused using 50, 100, 200, 300 gm-cm force by modified Allens' weight drop device. MEP and SSEP recordings were done before injury and at 0, 2, 4 and 6 hours after injury and on the 7th postoperative day. Neurological assessment was done at 24 hours and on the 7th day following injury. 50, 100, 200 gm-cm force caused partial injuries and 300 gm-cm force caused severe spinal cord injury. The predictive value of MEP and SSEP following partial injuries was 80% and 66.67% respectively. Both MEP and SSEP were 100% predictive in severe injury. MEP and SSEP monitoring can therefore be complementary to each other in predicting the neurological outcome in partial injuries to the spinal cord.

Surgery of perirolandic mass lesions with central sulcus mapping.

In a series of forty eight consecutive patients with parenchymatous mass lesions in the perirolandic area the central sulcus was identified intraoperatively in forty six. In patients in whom the mass lesion was seen on the surface, the relationship of the lesion to the somatosensory cortex and its underlying white matter was precisely determined. When the lesion was subcortical, the relationship of the mass lesion to the central sulcus on the MR image together with the central sulcus identified peroperatively, again, helped to determine the exact site of the tumour. Knowledge of the exact location of the tumour in relation to the central sulcus helped in the radical excision of seventy seven percent of these tumours. The morbidity following the operation was minimal.