In this Ph.D. thesis, possibilities to produce human proinsulin in its native conformation in the periplasm of E. coli as well as strategies for in vitro folding of proinsulin were analyzed. Production of native proinsulin in E. coli was optimized by means of different strategies (as fusion protein with DsbA, cosecretion of chaperones, variation of cultivation conditions and addition of thiol containing substances to the medium). Native proinsulin was detected using a conformation specific ELISA and a yield of 1 - 9 mg/g dry cell weight could be obtained. Besides the production of proinsulin in the native conformation in E. coli, in vitro refolding of denatured, reduced proinsulin was investigated and analyzed by reversed phase HPLC. At optimized conditions a yield of 60 - 70 % or 6 mg/ml of native proinsulin was obtained. Aggregation and disulfide bond formation are limitations during proinsulin folding as identified by folding of proinsulin in the presence of protein-disulfide isomerase (PDI). Both, aggregation and disulfide bond formation, are influenced by the isomerase and chaperone function of PDI resulting in a faster folding and higher refolding yield. Therefore, proinsulin is the first model protein shown that PDI influences its folding by both functions.